Novel single nucleotide polymorphisms and combinations of novel and known polymorphisms for determining the allele-specific expression of the igf2 gene

ABSTRACT

Single nucleotide polymorphisms and uses for determining the imprinting status of the Insulin Growth Factor-2 gene in a clinical specimen are described.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 13/791,336, filed Mar. 8, 2013, which is a divisional of U.S. patentapplication Ser. No. 12/672,066, filed May 19, 2010, now U.S. Pat. No.8,420,315, issued Apr. 16, 2013, which is the U.S. National Stage entryof International Application No. PCT/US2008/072356, filed Aug. 6, 2008,which claims benefit of priority to U.S. Provisional Patent ApplicationNo. 60/954,290, filed Aug. 6, 2007 and U.S. Provisional PatentApplication No. 60/988,715, filed Nov. 16, 2007, each of which areincorporated by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file SEQTXT_(—)86894-884092-002523US,created on Aug. 6, 2013, 33,313 bytes, machine format IBM-PC, MS-Windowsoperating system, is hereby incorporated by reference in its entiretyfor all purposes.

BACKGROUND OF THE INVENTION

The gene for insulin-like growth factor 2, or IGF2, is located in acluster of imprinted genes on human chromosome 11p15.5. Genomicimprinting is an important mechanism of gene regulation where one copyof the gene is normally expressed and the other copy is silenced throughan epigenetic mark of parental origin. IGF2 is normally maternallyimprinted in human tissues and therefore, expressed only from thepaternally inherited copy of the gene (DeChiara T M, et al. Cell 64,849-859 (1991); Rainier S, et al., Nature 362, 747-749 (1993); Ogawa, etal, Nature 362, 749-751 (1993)). Loss of imprinting of IGF2 (referred toas loss of imprinting, or LOI) has been strongly linked to severalcancer types (over 20 tumor types reviewed in Falls, et al. 1999, AJP154, 635-647). Furthermore, mounting evidence indicates that individualsdisplaying LOI of IGF2 may be at elevated risk for developing colorectalcancer (Kinochi et al., 1996, Cancer Letters 107, 105-108 (1996);Nishihara S. 2000, Int. Jour. Oncol. 17, 317-322; Cui H 1998, NatureMedicine 4-11, 1276-1280; Nakagawa H 2001, PNAS 98-2, 591-596). LOI ofIGF2 can be detected in normal tissues of cancer patients includingperipheral blood and normal colonic mucosa (Kinochi et al., 1996, CancerLetters 107, 105-108 (1996); Ogawa, et al, Nature Genetics 5, 408-412(1993); Cui H, Science 299, 1753 (2003)) and in the normal tissues ofpeople believed to be cancer free (Cui H, et al. Nature Medicine 4-11,1276-1280 (1998); Cui H, Science 299, 1753 (2003); Woodson K et al.,JNCI 96, 407-410 (2004); Cruz-Correa Met al., Gastroenterology 126,964-970 (2004)).

Several studies of peripheral blood of general populations report thatbetween 7-10% of people display loss of imprinting of IGF2 in colonicmucosa tissue. Three retrospective studies report that the odds ofcolorectal cancer patients displaying LOI of IGF2 in either peripheralblood or colonic mucosa are significantly higher (between 2-21 fold)than the odds of an age matched cancer free control group displayingLOI. These studies suggest that LOI of IGF2 may predispose otherwisehealthy individuals to colorectal cancer. Therefore, a risk test basedon the detection of LOI of IGF2 may have a future clinical benefit, (CuiH, et al. Nature Medicine 4-11, 1276-1280 (1998); Cui H, Science 14,1753-1755 (2003); Woodson K 2004, JNCI 96, 407-410; Cruz-Correa M,Gastroenterology 126, 964-970 (2004)). These studies show that peoplewith LOI of IGF2 (also referred to as the IGF2 biomarker) may be up to20 times more likely to develop colorectal cancer than individualswithout the IGF2 biomarker.

Detection of LOI of IGF2 is based on a quantitative allele specific geneexpression assay, where transcripts from both copies of the IGF2 geneare each quantified. The quantities are then compared to one another todetermine an allelic gene expression ratio, which is subsequentlycompared to a threshold value. If the concentration of the lesserabundant allele is “relatively similar” to the concentration of the moreabundant allele, then the IGF2 imprint is determined to be lost. If theconcentration of the lesser abundant allele is “relatively dissimilar”to the concentration of the more abundant allele, then the IGF2 imprintis determined to be present. One method of measuring the imprintingstatus of IGF2 in a sample is to first determine the genotype(s) of oneor more polymorphic sites in the transcribed region of the IGF2 gene.Heterozygous markers in the transcribed region of the gene provide forconvenient molecular handles by which the individual alleles of the IGF2gene can be distinguished from one another in a sample. RNAtranscription from each of the two copies of the IGF2 gene may beindependently measured with quantitative allele specific assays.Comparison of the amount of expression of one allele to the amount ofexpression of the other allele can therefore be made and the imprintingstatus of the IGF2 gene can be determined (see FIG. 2).

IGF2 has four promoters, each driving expression of alternativelyspliced transcripts, in a tissue specific manner (FIG. 1A). Exons 7, 8,and 9 are present in all transcripts, while exons 1-6 have been reportedto be expressed in a promoter specific fashion. Exon 9 includes a shortstretch of protein-coding sequence followed by a considerably longer 3′UTR. Polymorphic markers in exons 7, 8, and 9 are therefore useful inthe determination of IGF2 imprinting status by enabling the detection ofallele specific expression of IGF2 transcription driven from any of thefour IGF2 promoters.

Four allele-specific expression assays measuring IGF2 imprinting statusare known to those skilled in the art. Woodson, et al. measuredimprinting status of IGF2 with a combination of two SNP based assays(rs680—analogous to SEQ ID NO: 64 in Table 1A; and rs2230949—analogousto SEQ ID NO: 56 in Table 1A) (Woodson K 2004, JNCI 96, 407-410). BothSNPs are in exon 9 of IGF2 but are reported by Woodson et al. to be inminimal linkage disequilibrium. Therefore attempts to measure LOI of anindividual with such a combination of markers increases the probabilitythat the individual will be heterozygous for at least one of the twoSNPs, and thereby increase the likelihood that the LOI status of theindividual can be determined. The authors demonstrated that the firstSNP, the second SNP, or both SNPs were informative (i.e., wereheterozygous and, therefore, permitted measurement of LOI of IGF2) in 48of 106 patients evaluated (or 45%). Cui et al. measured IGF2 imprintingwith a combination of two assays, one targeting a SNP (rs680—analogousto SEQ ID NO: 64 in Table 1A) and a second measuring restrictionfragment length polymorphisms of a simple sequence repeat within exon 9of IGF2. The authors demonstrated that the SNP, the repeat, or bothmarkers were informative in 191 of 421 (or 45%) patients evaluated (CuiH, et al. Nature Medicine 4-11, 1276-1280 (1998)).

Previous studies have demonstrated that use of these polymorphismsresult in a low combined frequency of heterozygosity in patientpopulations and, therefore, a large number of individuals in thesepopulations were “uninformative” such that their IGF2 imprinting statuscould not be determined. The present application describes newlydiscovered SNPs in IGF2 exon 9, and the discovery of useful combinationsof SNPs, which enable successful LOI measurements in an increasedproportion of the human population. The ability to measure LOI usingthese polymorphisms in the general population will have a profoundmedical benefit, serving as the basis for various molecular diagnosticand therapeutic tests.

The informativity of a given SNP for detection of LOI is based on thefrequency of heterozygosity of the SNP within a population. Furthermore,the optimal informativity of a combination of different SNPs isdependent upon the linkage among the different markers. For example, iftwo SNPs fall within a common haplotype block, the combined use of thetwo SNPs provides a minimal increase in informativity relative to theuse of either of the two SNPs alone. However, if two SNPs are not on thesame haplotype block (i.e., are in minimal linkage disequilibrium), thecombined use of the two SNPs provides an effective increase ininformativity relative to the use of either of the two SNPs alone.

The recent release of the HapMap II human genetic variation datasetprovides haplotype analysis of genome-wide DNA sequence data. In theHapMap II study, SNPs were identified in 270 people genotyped from fourgeographically diverse populations, including 30 mother-father-adultchild trios from the Yoruba in Ibadan, Nigeria; 30 such trios ofnorthern and western European ancestry living in Utah; unrelated HanChinese individuals in Beijing and 45 unrelated Japanese individuals inTokyo. Haplotype analysis of those SNPs within an approximately 70 Kbregion including the IGF2 locus provides a view of haplotype blockspredicted by this current and extensive dataset. In FIG. 3, theHaploview visualization of linkage prediction is depicted below ato-scale diagram of the IGF2 locus. Three haplotype blocks areidentified (represented as black horizontal bars positioned in scalewith the IGF2 locus). The data predict haplotype blocks spanning fromapproximately 14 to 19 Kb upstream of IGF2 exon 1, from approximately 1Kb upstream of exon 3 to approximately 5 Kb downstream of exon 4, andfrom approximately 2 Kb upstream of the start of exon 9 to approximately14 Kb downstream of the end of exon 9, a haplotype block thatencompasses exons 8 and 9. In general, regions between these haplotypeblocks display minimal linkage disequilibrium and provide strongevidence for historic recombination (indicated by white diamondsrepresenting multiple pairwise SNP comparisons). These data aresummarized in FIG. 1B. Haplotype blocks are represented by blackhorizontal bars and the region of predicted minimal linkagedisequilibrium is represented by a grey horizontal bar.

Gaunt et al. performed an association studying for body mass index (BMI)in a Caucasian cohort of 2,734 European men using 12 SNPs ranging fromjust upstream of IGF2 exon 1 to approximately 1 Kb prior to the end ofthe exon 9 3′ UTR, (Gaunt et al. Human Mol Genet. vol. 10, no. 14:1491-1501). This study included linkage analysis of a single SNP(rs680—analogous to SEQ ID NO: 64 in Table 1A), which had one allelereported to be positively associated with high BMI in the cohort, toeach of the other 11 SNPs in a pair wise fashion. The authors report ahaplotype block within the 3′ UTR of exon 9, containing 3 SNPs fromtheir study (see Example 3 the black horizontal bar in FIG. 1C, and thegrey bar in FIG. 4).

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of determining a SNP genotype ina human individual. In some embodiments, the methods comprisedetermining, in a sample containing genomic DNA from the individual, thenucleotide or nucleotides at the polymorphic nucleotide of a singlenucleotide polymorphism (SNP), wherein the SNP is selected from thegroup consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47.In some embodiments, the nucleotide at the polymorphic position of theSNP (and therefore at the corresponding position of the polynucleotide)is an underlined nucleotide as displayed in Table 2A or 2B.

The present invention also provides methods of quantifyingallelic-specific expression of RNA in a human individual, wherein thehuman individual is a heterozygote for a single nucleotide polymorphism(SNP) in the Insulin Growth Factor-2 (IGF2) gene. In some embodiments,the methods comprise quantifying the amount of RNA in a sample from thehuman individual comprising one or each polymorphic option of the SNP,wherein the SNP is selected from the group consisting of SEQ ID NO: 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, and 47.

In some embodiments, the sample is a blood or tissue sample. In someembodiments, the sample is a stool sample. In some embodiments, themethods further comprise correlating the relative amount of RNAcomprising each polymorphic option of the SNP to loss of imprinting ofthe IGF2 gene. In some embodiments, the correlating step comprisescorrelating the relative amount of RNA to a prognosis or diagnosis ofcancer or a prediction of efficacy of a drug for treating cancer. Insome embodiments, the RNA is reverse transcribed into cDNA and thequantity of allele specific cDNA is used to determine the amount of RNA.

In some embodiments, the methods further comprise determining whetherthe individual is homozygous or heterozygous for one or more SNPs.

The present invention also provides isolated polynucleotides of between8-100 nucleotides, wherein the polynucleotide distinguishes between oneallele of a SNP (or complement thereof) and the other allele of the SNP(or complement thereof) in a hybridization reaction, wherein the SNP isselected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, and 47.

In some embodiments, the penultimate or ultimate 3′ nucleotide of thepolynucleotide hybridizes to the polymorphic nucleotide of the SNP.

The present invention also provides isolated polynucleotides of between8-100 nucleotides wherein the polynucleotide functions as a primer inInsulin-like Growth Factor 2 (IGF2) cDNA, such that the polynucleotidehybridizes to the cDNA and the 3′ nucleotide of the polynucleotide iscomplementary to the nucleotide immediately upstream of the polymorphicnucleotide of a SNP, wherein the SNP is selected from the groupconsisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47.

In some embodiments, at least the 10 contiguous 3′ nucleotides of thepolynucleotide are complementary to the cDNA.

The present invention also provides isolated polynucleotides comprisinga SNP sequence, or complement thereof, selected from the groupconsisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47, whereinthe nucleotide at the polymorphic position of the SNP is an underlinednucleotide as displayed in Table 2A or 2B.

The present invention also provides kits comprising an isolatedpolynucleotide:

of between 8-100 nucleotides wherein the polynucleotide functions as aprimer in Insulin-like Growth Factor 2 (IGF2) cDNA, such that thepolynucleotide hybridizes to the cDNA and the 3′ nucleotide of thepolynucleotide is complementary to the nucleotide immediately upstreamof the polymorphic nucleotide of a SNP; orof between 8-100 nucleotides wherein the polynucleotide functions as aprimer in Insulin-like Growth Factor 2 (IGF2) cDNA, such that thepolynucleotide hybridizes to the cDNA and the 3′ nucleotide of thepolynucleotide is complementary to the nucleotide immediately upstreamof the polymorphic nucleotide of a SNP, wherein the SNP is selected fromthe group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and47.

In some embodiments, the kit comprises a first isolated polynucleotideof between 8-100 nucleotides, wherein the polynucleotide distinguishesbetween a first allele of a SNP and a second allele of the SNP (orcomplement thereof) in a hybridization reaction.

In some embodiments, the kit further comprises a second isolatedpolynucleotide of between 8-100 nucleotides, wherein the polynucleotidedistinguishes between the first allele of the SNP (or complementthereof) and the second allele of the SNP (or complement thereof), andwherein the first polynucleotide is complementary to the polymorphicnucleotide in the first allele and the second polynucleotide iscomplementary to the polymorphic nucleotide of the second allele.

In some embodiments, the kit further comprises one or more primer foramplifying a region of the IGF2 locus encompassing the polymorphic site,wherein the one or more primer is different from the isolatedpolynucleotide.

In some embodiments, the kit further comprises a DNA polymerase. In someembodiments, the polymerase is a thermostable DNA polymerase. In someembodiments, the kit further comprises a reverse transcriptase. In someembodiments, the first and/or second polynucleotide is detectablylabeled.

The present invention also provides reaction mixture comprising anisolated polynucleotide: of between 8-100 nucleotides wherein thepolynucleotide functions as a primer in Insulin-like Growth Factor 2(IGF2) cDNA, such that the polynucleotide hybridizes to the cDNA and the3′ terminal nucleotide of the polynucleotide is complementary to thenucleotide immediately upstream of the polymorphic nucleotide of a SNPso that extension of the polynucleotide by a polymerase incorporates anucleotide complimentary to the polymorphic nucleotide of the SNP; or

of between 8-100 nucleotides wherein the polynucleotide functions as aprimer in Insulin-like Growth Factor 2 (IGF2) cDNA, such that thepolynucleotide hybridizes to the cDNA and the 3′ nucleotide of thepolynucleotide is complementary to the nucleotide immediately upstreamof the polymorphic nucleotide of a SNP so that extension of thepolynucleotide by a polymerase incorporates a nucleotide complimentaryto the polymorphic nucleotide of the SNP, wherein the SNP is selectedfrom the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,and 47.

In some embodiments, the reaction mixtures comprise:

a first isolated polynucleotide of between 8-100 nucleotides, whereinthe polynucleotide distinguishes between a first allele of a SNP (or acomplement thereof) and a second allele of the SNP (or a complementthereof) in a hybridization reaction; a thermostable DNA polymerase; andhuman genomic DNA or cDNA from reverse-transcription of human RNA.

In some embodiments, the first isolated polynucleotide hybridizes to theDNA. In some embodiments, the polymerase is a thermostable DNApolymerase.

In some embodiments, the reaction mixtures further comprise a secondisolated polynucleotide of between 8-100 nucleotides, wherein thepolynucleotide distinguishes between the first allele of the SNP (orcomplement thereof) and the second allele of the SNP (or complementthereof), and wherein the first polynucleotide is complementary to thepolymorphic nucleotide in the first allele and the second polynucleotideis complementary to the polymorphic nucleotide of the second allele.

In some embodiments, the reaction mixtures further comprise one or moreprimer for amplifying a region of the IGF2 locus encompassing thepolymorphic site, wherein the one or more primer is different from theisolated polynucleotide.

The present invention provides methods of quantifying allelic-specificexpression of RNA in a human individual, wherein the human individual isa heterozygote for at least two single nucleotide polymorphisms (SNPs)in the Insulin Growth Factor-2 (IGF2) gene. In some embodiments, themethods comprise:

quantifying the amount of RNA in a sample from the human individualcomprising one or more polymorphic option of each of at least two SNPs,wherein the two SNPs are each selected from “Linkage Block” 1 of Tables1A, 1B, 1C, 2A or 2B; orquantifying the amount of RNA in a sample from the human individualcomprising one or each polymorphic option of each of at least two SNPs,wherein the at least two SNPs are each selected from “Linkage Block” 2of Tables 1A, 1B, 1C, 2A or 2B; orquantifying the amount of RNA in a sample from the human individualcomprising one or each polymorphic option of each of at least two SNPs,wherein the at least two SNPs are each selected from “Linkage Block” 3of Tables 1A, 1B, 1C, 2A or 2B.

In some embodiments, the sample is a blood or tissue sample. In someembodiments, the sample is a stool sample. In some embodiments, themethod further comprises correlating the relative amount of RNAcomprising each polymorphic option of the SNPs to loss of imprinting ofthe IGF2 gene and/or predisposition for cancer.

In some embodiments, the RNA is reverse transcribed into cDNA and thequantity of allele-specific cDNA is used to determine the amount of RNA.

DEFINITIONS

A “thermostable polymerase” refers to a polymerase useful for PCRapplications. A thermostable polymerase can generally be heated to 75°C. repeatedly (e.g., at least 20 times for a minute each time) andretain at least 80% of its original activity. Examples of suchpolymerases include, but are not limited to, Taq polymerase.

A “single nucleotide polymorphism” or “SNP” refers to a site of onenucleotide that varies between alleles.

An “allele” refers to one member of a pair or set of different forms ofa gene. In a diploid organism, an individual has two copies of eachautosomal gene. For a single nucleotide polymorphism, an individual hastwo different alleles of the polymorphic nucleotide if the genotype atthe polymorphic nucleotide is different on one copy of the gene than theother copy of the gene (i.e. the individual is heterozygous for thepolymorphic nucleotide). If an individual has the same genotype at thepolymorphic nucleotide on both copies of the gene (i.e. the individualis homozygous for the polymorphic nucleotide), then the individual hastwo copies of the same allele of the polymorphic nucleotide. A givenindividual can be homozygous for one polymorphic nucleotide within agene (two copies of the same allele of the polymorphic nucleotide) andheterozygous for a different polymorphic nucleotide within the same gene(two different alleles of the polymorphic nucleotide).

“Hybridization” refers to the formation of a duplex structure by twosingle stranded nucleic acids due to complementary base pairing.Hybridization can occur between exactly complementary nucleic acidstrands or between nucleic acid strands that contain minor regions ofmismatch.

“Target sequence” or “target region” refers to a region of a nucleicacid that is to be analyzed and comprises the polymorphic site ofinterest.

As used herein, the terms “nucleic acid,” “polynucleotide” and“oligonucleotide” refer to nucleic acid regions, nucleic acid segments,primers, probes, amplicons and oligomer fragments. The terms are notlimited by length and are generic to linear polymers ofpolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), and any other N-glycoside ofa purine or pyrimidine base, or modified purine or pyrimidine bases.These terms include double- and single-stranded DNA, as well as double-and single-stranded RNA.

A nucleic acid, polynucleotide or oligonucleotide can comprise, forexample, phosphodiester linkages or modified linkages including, but notlimited to phosphotriester, phosphoramidate, siloxane, carbonate,carboxymethylester, acetamidate, carbamate, thioether, bridgedphosphoramidate, bridged methylene phosphonate, phosphorothioate,methylphosphonate, phosphorodithioate, bridged phosphorothioate orsulfone linkages, and combinations of such linkages.

A nucleic acid, polynucleotide or oligonucleotide can comprise the fivebiologically occurring bases (adenine, guanine, thymine, cytosine anduracil) and/or bases other than the five biologically occurring bases.For example, a polynucleotide of the invention can contain one or moremodified, non-standard, or derivatized base moieties, including, but notlimited to, N⁶-methyl-adenine, N⁶-tert-butyl-benzyl-adenine, imidazole,substituted imidazoles, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxymethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, uracil-5-oxyaceticacidmethylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,2,6-diaminopurine, and 5-propynyl pyrimidine. Other examples ofmodified, non-standard, or derivatized base moieties may be found inU.S. Pat. Nos. 6,001,611; 5,955,589; 5,844,106; 5,789,562; 5,750,343;5,728,525; and 5,679,785.

Furthermore, a nucleic acid, polynucleotide or oligonucleotide cancomprise one or more modified sugar moieties including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and a hexose.

“Haplotype block” refers to a region of a chromosome that contains oneor more polymorphic sites (e.g., 1-10) that tend to be inheritedtogether. In other words, combinations of polymorphic forms at thepolymorphic sites within a block cosegregate in a population morefrequently than combinations of polymorphic sites that occur indifferent haplotype blocks. Polymorphic sites within a haplotype blocktend to be in linkage disequilibrium with each other. Often, thepolymorphic sites that define a haplotype block are common polymorphicsites. Some haplotype blocks contain a polymorphic site that does notcosegregate with adjacent polymorphic sites in a population ofindividuals.

“Linkage disequilibrium” refers to the preferential segregation of aparticular polymorphic form with another polymorphic form at a differentchromosomal location more frequently than expected by chance. Linkagedisequilibrium can also refer to a situation in which a phenotypic traitdisplays preferential segregation with a particular polymorphic form oranother phenotypic trait more frequently than expected by chance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the structure of the IGF2 gene. The scale bar abovethe gene diagram is drawn in 1 Kb increments and depicts the location ofIGF2 on chromosome 11 (NCBI build 36). The scale bar is also present inFIG. 4. Arrows in FIG. 1A, represent the four promoters of IGF2. Exons 1to 9 are indicated below the gene diagram. Black shaded exons 1 to 6 arenot protein-coding in most transcripts of IGF2. The white exons 7 and 8are protein-coding, as is the small white region of exon 9. The blackshaded region of exon 9 is the 3′ UTR.

FIG. 1B illustrates the regions of IGF2 predicted to fall withinhaplotype blocks (black horizontal bars) and within regions predicted tobe in low linkage disequilibrium (grey horizontal bar) based onhaplotype analysis of HapMap II genotyping data, as described in theBackground section.

FIG. 1C illustrates the region of IGF2 predicted to fall within ahaplotype block (black horizontal bars) and within two uncharacterizedregions of IGF2 (grey horizontal bars) based on a previous study (Gauntet al. Human Mol Genet. 10(14):1491-1501 (2001)), as described in theBackground section and in Example 3.

FIG. 2 illustrates a basic strategy for determining LOI of IGF2 in abiological sample. Genomic DNA and total or polyadenylated RNA areisolated from a biological sample (for example, peripheral blood,peripheral blood mononuclear cells, stool, etc.) from and individual.The genomic DNA sample is used for genotyping of one or more polymorphicmarkers (DNA SNP Assay). This step determines whether the individual isheterozygous for a specific SNP or combination of SNPs. Any SNP, orcombination of SNPs, determined to be heterozygous may be utilized foranalysis of allele-specific expression of the IGF2 gene in the matchedRNA sample (RNA SNP assay). In the example shown, an individual wasdetermined by an allele-discriminating DNA genotyping assay to behomozygous for hypothetical SNPs 1, 3, 4 and 6 and heterozygous forhypothetical SNPs 2 and 5. cDNA is amplified from the relevant region ofthe IGF2 transcript using standard reverse transcriptase and PCR methodssuch that PCR products including at least SNPs 2 and 5 are amplified.Expression from each of the two copies of the IGF2 gene is independentlymeasured using the generated cDNA with a quantitative allele specificgene expression assay, and that can sufficiently discriminate betweenthe two alleles of the gene. Comparison of the amount of expression ofone allele relative to the amount of expression of the other allelethereby determines the imprinting status of the IGF2 gene. Assays thatdiscriminate SNPs 2 and 5 may be performed simultaneously, and allelespecific expression ratios obtained for each assay can be compared toimprove accuracy.

FIG. 3 illustrates the haplotype analysis of HapMap II SNP genotypedata, as described in the Background section. The gene diagram of IGF2(described in FIG. 1A) is shown, with Haploview-generated linkage datarepresented below. Vertical lines indicate the positions of predictedhaplotype blocks (horizontal black bars) relative to the IGF2 genediagram. Diamonds are shaded based on the linkage data for the indicatedpair-wise comparison of SNPs. Black diamonds represent SNP pairs forwhich there is strong evidence for linkage disequilibrium (D′=1 and LODscore≧2). White diamonds represent SNP pairs for which there is strongevidence for historical recombination, indicating minimal linkagedisequilibrium (D′<1 and LOD score<2). Grey diamonds represent SNP pairswhich provided uninformative data for determination of linkagedisequilibrium. dbSNP identifiers for the SNPs defining the boundariesof the predicted haplotype blocks are indicated at the bottom left ofthe figure. Analysis of HapMap II data supports a haplotype blockencompassing exon 9.

FIG. 4 illustrates the haplotype analysis of SNP genotype data for acombined cohort of Caucasian, African American, Chinese, Japanese andMexican individuals generated by the study described in the presentapplication. The gene diagram of IGF2 is shown. Vertical lines indicatethe relative positions of analyzed SNPs, and extend to the Haploviewvisualization of linkage analysis below. The horizontal grey barrepresents the haplotype block, and the horizontal white bars representthe uncharacterized regions of exon 9 as determined by Gaunt et al. (seeBackground section and Example 3). Shading of diamonds is as describedin FIG. 3. The SEQ ID NO: numbers for analyzed SNPs and the previouslyreported linkage block (Gaunt Block) to which each belongs are indicatedat the bottom left.

FIG. 5 illustrates the use of a restriction enzyme based assay forgenotyping the SNP corresponding to SEQ ID NO: 64. The polymorphicnucleotide is located within the recognition sequence of two restrictionenzymes. Apa I recognizes and cleaves the sequence when the “G” alleleis present, and Ava II recognizes and cleaves the sequence when the “A”allele is present. A PCR amplicon including SEQ ID NO: 64 was amplifiedfrom three independent genomic DNA samples derived from threeindividuals (Samples A, B and C). Amplicons were digested with Apa I orAva II or a combination of both enzymes (Double). Digestion by Apa Ionly indicates that the individual is homozygous for the G allele(Sample B), digestion by Ava II only indicates that the individual ishomozygous for the A allele (Sample C), and digestion by both enzymesindicates that the individual is heterozygous for the SNP (Sample A).

FIG. 6 illustrates the use of a restriction enzyme based method fordetecting LOI of IGF2. Total RNA was extracted from three individualsthat are heterozygous for SEQ ID NO:64. The region including SEQ IDNO:64 was RT-PCR amplified from each sample. cDNA amplicons weredigested with Apa I or Ava II or a combination of both enzymes, asindicated above each lane. Digested products were resolved on an AgilentBioanalyzer, and concentrations of cut and uncut fragments weredetermined. The quantity of fragments cut by Apa I represents theproportion of cDNA including the “G” allele. The quantity of fragmentscut by Ava II represents the proportion of cDNA including the “A”allele. Therefore, the ratio of Apa I cut fragments to Ava II cutfragments indicates the relative ratio of expression of the two allelesin the original RNA sample. The calculated G:A ratio is shown below eachtriplet of lanes representing each sample. Sample 2 expressesexclusively the “A” allele. Samples 1 and 3 express both alleles (i.e.display LOI IGF2), with G:A ratios of 0.5 and 0.3, respectively.

FIG. 7 diagrams a method for allele-specific detection of a SNP using asingle nucleotide primer extension strategy. The SNP represented by SEQID NO:64 and its surrounding DNA sequence are shown as an example (“PCRamplicon sequence”—SEQ ID NO:114). The nucleotide position of SEQ IDNO:64 is indicated by the arrow labeled “SeqID 64”. The sequence of thepolynucleotide used for single nucleotide primer extension is indicatedby the bracket labeled “Primer” (SEQ ID NO:113). The PCR DNA amplicon(or, alternatively a RT-PCR cDNA amplicon) including the sequence ofinterest is amplified from the sample to be assayed. A primer is addedto the purified PCR (or, alternatively RT-PCR) product that anneals withits 3′ terminal nucleotide complimentary to the template nucleotide 1base to the 3′ side of the polymorphic nucleotide to be genotyped.Single nucleotide primer extension is carried out using a thermostableDNA polymerase and differentially fluorescently labeled ddNTPs. In thisexample, either dR110-labeled ddGTP or dR6G-labeled ddATP is added tothe 3′ end of the primer (generating SEQ ID NOs: 115 and 116,respectively). These labeled polynucleotides are then resolved and thepeak areas representative of each possible incorporated nucleotide arecalculated. Peak areas are compared to determine the genotype of theindividual at that SNP position (or, alternatively the allele-specificgene expression ratio).

FIG. 8 illustrates the use of the single nucleotide primer extensionassay described in FIG. 7 to genotype three individuals for SEQ IDNO:64. The three individuals that were assayed for LOI of IGF2 by therestriction enzyme based assay shown in FIG. 6 were genotyped. Thefigure shows the resulting chromatograms for each sample followingresolution and peak detection using an ABI Genetic Analyzer and GeneMapper software. As expected, peaks representing both alleles of SEQ IDNO:64 are obtained in relatively equal proportions, confirming that thethree individuals are heterozygous for SEQ ID NO:64 and demonstratingthe concordance between results obtained by the restriction enzyme basedmethod and the single nucleotide primer extension based method.

FIG. 9 illustrates the application of the single nucleotide primerextension based method for detecting LOI of IGF2. The region includingSEQ ID NO:64 was RT-PCR amplified from a total RNA sample derived fromeach of the three individuals genotyped in FIG. 8. The cDNA productsobtained were purified and analyzed as diagrammed in FIG. 7. The figureshows the resulting chromatograms for each sample following resolutionand peak detection using an ABI Genetic Analyzer and Gene Mappersoftware. For each sample, peak areas representing each of the twopossible alleles were calculated and compared to each other. Thecalculated G:A ratios are indicated to the right of each chromatogram.Consistent with the results shown in FIG. 6, Samples 1 and 3 weredetermined to show LOI of IGF2, and Sample 2 was determined to shownormal imprinting of IGF2.

FIG. 10 illustrates the quantitative analytical linearity of singlenucleotide primer extension assays developed for nine independent SNPs.The SEQ ID number for each assayed SNP is indicated to the right of thegraph. For each assay, PCR products were separately amplified fromgenomic DNA samples derived from two individuals; one homozygous for oneallele of the SNP and the other homozygous for the other allele of theSNP. The PCR products were purified and quantified. For each of the nineSNPs, two PCR products (one amplified from the DNA sample homozygous forone allele and the other amplified from the DNA sample homozygous forthe other allele) were combined in the following ratios of allele 1 toallele 2; 1:10, 1:8, 1:6, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 6:1, 8:1and 10:1. For each of the nine SNPs, the single nucleotide primerextension assay was performed in triplicate on each dilution point. Peakareas representing each of the two possible alleles were calculated(y-axis) and compared to the known input ratio of each allele pair(x-axis). Values are plotted on a log 10 scale. The average R² of theassays is 0.996±0.002 and the average slope is 0.830±0.024,demonstrating that each assay is capable of sensitively and accuratelymeasuring relative quantitative ratios of each allele pair representedin a sample.

DETAILED DESCRIPTION I. Introduction

The present invention provides methods of detecting LOI of IGF2 andincludes novel single nucleotide polymorphism (SNPs) in the IGF2 gene.Detection of these SNPs, alone or in combination with each other, or incombination with previously known SNPs, provide a valuable new way todetect, for example, loss of imprinting of IGF2. The new SNPs can, aloneor in combination, be used to independently monitor expression of eachof a human individual's two copies of the IGF2 gene, and can be used todetermine the imprinting status of IGF2 in a biological sample. Forexample, if an individual is heterozygous for a particular IGF2 SNP,then probes or other reagents can be employed to separately detect andquantify RNA from each IGF2 allele. If one allele is predominantlyexpressed, then imprinting of IGF2 is likely occurring. However, if bothalleles are expressed at similar levels, it is likely that loss ofimprinting of IGF2 has occurred.

Further, it is now possible to monitor loss of imprinting in many morehuman genetic and racial backgrounds. As an example, the presentinvention provides a number of SNPs that commonly occur in AfricanAmerican, Caucasian, Chinese, Japanese and Mexican populations, therebyallowing for more useful methods for determining cancer risk in thosepopulations than ever before.

In addition to the discovery of novel IGF2 SNPs, the present inventionalso provides for combinations of IGF2 SNPs that provide a surprisingimprovement in the ability to detect LOI in individuals compared to whatwas predicted previously. For example, prior research into geneticrecombination frequency at the IGF2 locus described the existence ofblocks of low recombination, indicating that there would be no advantagefor using two or more SNPs within the same block. See, e.g., HapMap II(NCBI build 36); Gaunt et al., supra. These blocks are depicted in FIGS.1B and 1C. However, as shown in the data presented herein (see, e.g.,FIG. 4), there is in fact substantial recombination within these“blocks” and therefore detection of two or more SNPs within these“blocks” provides a substantial improvement in the ability to detect LOIin individuals than was predicted in the prior art.

Accordingly, the invention provides for the combination of SNPs (eitheras first described herein or as previously known) that are surprisinglyeffective in improving the accuracy of LOI determination as well asexpanding the possible populations of people for which the assay will beeffective (where a person is heterozygous for at least one SNP).

II. IGF2 SNPs

The following sequence identifiers represent SNP sequences within theIGF2 locus selected from the group consisting of SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, and 112.

In one embodiment, the invention provides isolated nucleic acids thatcomprise at least one SNP having one or the other polymorphic sequence,wherein the SNP sequences are selected from the group consisting of SEQID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47.

The present invention provides polynucleotides that distinguish betweentwo alleles of a SNP, wherein the SNP is selected from the groupconsisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47.

For example, the present invention provides polynucleotides thathybridize to a first allele of a particular SNP, but does notsignificantly hybridize to the second allele of the SNP. “Does notsignificantly hybridize” means that in the presence of equal amounts ofboth alleles in a sample, the polynucleotide is able to detect thepresence of the first allele but does not detect the presence of thesecond allele to such an extent so as to interfere with theinterpretation of the assay. In some embodiments, in the presence ofequal amounts of both alleles in a sample, the polynucleotide provides asignal for a sample having the first allele that is at least, e.g.,about 100; 1,000; 10,000; 100,000 times or more than the signalgenerated by the polynucleotide for a sample having an equal amount ofthe second allele. “Signal” refers to any output indicative ofhybridization of the polynucleotide to a complementary sequence. In someembodiments, at least 70%, 80%, 90%, 95% of the sequence of thepolynucleotide is complementary to a SNP selected from SEQ ID NO:s1-112, for example, they have at least 8, 10, 15, 20, 30, 40, 50complementary nucleotides.

Alternatively, the polynucleotides can distinguish between two allelesof a SNP by acting as a primer in a template-specific primer extensionreaction. In these embodiments, the polynucleotides do not generallyencompass the polymorphic nucleotide but instead hybridize to thegenomic DNA such that 3′ extension of the polynucleotide occurs at thepolymorphic nucleotide. Thus, in some embodiments, the 3′ end of thepolynucleotide is complementary to a nucleotide within 10, 5, 3, 2, or 1nucleotide(s) upstream from the polymorphic nucleotide. In someembodiments, the polynucleotides are complementary over at least 70%,80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the polynucleotides lengthto an IGF2 cDNA. In some embodiments, the polynucleotide comprises atits 3′ end, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or morecontiguous nucleotides that are at least at least 70%, 80%, 90%, 95%,96%, 97%, 98%, 99%, or 100% complementary to an IGF2 cDNA. Optionally,the 5′ end of the polynucleotide will comprise a sequence tag or othersequence not complementary to an IGF2 cDNA. As is well known in the art,a variety of primer extension methods can be employed to detect SNPs.

In some embodiments, the polynucleotides that distinguish between thetwo alleles are at least 4, 6, 8, 10, 12, 15, 20, 30, 50, or morenucleotides in length. In some embodiments, the polynucleotides are nomore than 1000, 500, 200, 100, 80, 50, 40, 30, or 25 nucleotides inlength. For example, the polynucleotides can be, e.g., 8-25, 8-30, 8-50,8-100, 10-25, 10-50, 10-100, nucleotides, etc. The polynucleotides thatdistinguish between the two alleles will typically include a nucleotidethat corresponds (i.e., aligns with) and is complementary to one of thepolymorphic nucleotides of the SNP. In some embodiments, the ultimate orpenultimate 3′ nucleotide of the polynucleotide is complementary to anucleotide at the polymorphic position of the SNP. Such embodiments canbe particularly useful in SNP detection methods employing thepolynucleotides as primers or probes, for example in amplification-basedassays such as those involving the polymerase chain reaction.

The polynucleotides of the invention can be detectably labeled.Detectable labels suitable for use in the present invention include anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include biotin for staining with labeledstreptavidin conjugate, magnetic beads (e.g., Dynabeads™), fluorescentdyes (e.g., fluorescein, Texas red, rhodamine, green fluorescentprotein, and the like, see, e.g., Molecular Probes, Eugene, Oreg., USA),radiolabels (e.g., ³H, ¹²⁵H, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in anELISA), and colorimetric labels such as colloidal gold (e.g., goldparticles in the 40-80 nm diameter size range scatter green light withhigh efficiency) or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads. Patents teaching the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241.

Hybridization reaction conditions can vary depending on the assay thatis used to detect the SNPs. Stringent, sequence-specific hybridizationconditions, under which an oligonucleotide will hybridize only to theexactly complementary target sequence, are well known in the art.Stringent conditions are sequence dependent and will be different indifferent circumstances. Generally, stringent conditions are selected tobe about 5° C. lower than the thermal melting point (Tm) for thespecific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength and pH) at which 50% of thebase pairs have dissociated. Relaxing the stringency of the hybridizingconditions will allow sequence mismatches to be tolerated; the degree ofmismatch tolerated can be controlled by suitable adjustment of thehybridization conditions.

For Southern-type hybridization, exemplary conditions are: 50%formamide, 5×SSC, and 1% SDS, incubating at 42° C., or 5×SSC, 1% SDS,incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 55° C., 60°C., or 65° C. Such washes can be performed for 5, 15, 30, 60, 120, ormore minutes. For PCR applications (involving hybridization and/orextension of primers and/or probes), hybridization conditions comprisingannealing and extension condition are well known, e.g., as described inPCR Protocols: A Guide to Methods and Applications (Innis et al., eds.,1990).

The present invention relies on routine techniques in the field ofrecombinant genetics. Basic texts disclosing the general methods of usein this invention include Sambrook et al., Molecular Cloning, ALaboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994)).

III. Methods for Measuring Loss of Imprinting and Cancer Predisposition

Detection of LOI is based on a comparison of the amount of expressionderived from each of the two copies of the IGF2 gene within a biologicalsample from an individual. Thus, if an individual has two differentalleles of the IGF2 gene, then allele-specific detection can be used toquantify expression of each copy of the gene. If imprinting isfunctioning, then one copy of the gene (typically the maternal copy)will not be expressed in spite of the presence of a genomic copy of thegene. However, if LOI has occurred, then expression will occur from boththe maternal and paternal copies of the IGF2 gene. Because expressionlevels are not always exactly equal when LOI has occurred, in someembodiments, a sample is determined to display LOI of IGF2 if thequantified proportion of the lesser abundant allele is greater than orequal to 33.3% the quantified proportion of the more abundant allele.

It is generally desirable to know whether an individual is heterozygousfor a particular SNP. Thus in some embodiments, both DNA (i.e., genomicDNA) and RNA from a sample are obtained. The genomic DNA is assayed todetermine whether the individual is heterozygous for a particular SNP.If the individual is heterozygous, then it is possible to measure lossof imprinting by detecting RNA having either of the two SNP alleles andthen comparing their expression. This is illustrated in FIG. 2. In somecircumstances, however, it may be beneficial to detect only RNA withoutknowing whether the individual is a heterozygote for a particular SNP.In this circumstance, observing expression of two alleles indicates LOI,while detecting expression of one allele is not informative because itwill not be known if the negative result is due to imprinting orhomozygosity of the particular SNP. However, by increasing the number ofdifferent SNPs detected, it is possible to design an assay such that thechance of an individual being homozygous for every SNP would beextremely low.

In some embodiments, more than one SNP is assayed for an individual.“Assayed” or “assayed for” refers to separately quantifying eachpossible allele of the SNP. Generally, any combination of SNPs can beassayed for in a sample. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 75, 100, or more different SNPs are assayed todetermine whether LOI of IGF2 has occurred. Optimally, amplicons aredesigned that encompass more than one SNP, thereby allowing forefficient detection of multiple SNPs.

As explained in detail in Example 3, one novel feature of the presentinvention is the detection of two SNPs in general proximity to eachother improves both accuracy of the assay as well as the number ofpossible heterozygous candidates. This latter finding is particularlysurprising in view of earlier reports implying that certain genomicregions segregate as linkage blocks. In view of the discoveriesdescribed in Example 3, another novel feature of the present inventionis the detection of the relative amounts of the polymorphic options oftwo or more SNPs, wherein each SNP is selected from the same “Block” aslisted in Tables 1A, 1B, 1C, 2A and 2B. Thus, for example, two or moreSNPs are assayed for in Block 1. Or, two or more SNPs are assayed for inBlock 2. Or, two or more SNPs are assayed for in Block 3. These optionsdo not preclude further addition of SNPs from other Blocks. Simply as anexample, this means that two SNPs from Block 1 and one SNP from Block 2could be assayed for.

As shown in Tables 4-8, various racial groups display differentoccurrence rates of heterozygosity for the SNPs. Thus, in someembodiments, SNPs are selected for use within a particular racial groupto allow for improved chance of assaying for SNPs that are heterozygousin a particular racial group. Thus, in some embodiments, one or moreSNPs in Table 4 are assayed for in people of Chinese descent, one ormore SNPs in Table 5 are assayed for in people of Japanese descent, oneor more SNPs in Tables 6 are assayed for in people of African descent,one or more SNPs in Table 7 are assayed for in Caucasian people, and oneor more SNPs in Table 8 are assayed for in people of Mexican descent.

Alternatively, one set of SNPs can be selected to allow for the greatestchance of assaying for a heterozygous SNP regardless of race. Thus, insome embodiments, a panel of SNPs selected from Tables 4-8 are used.

In further embodiments, a person of a certain racial group as listed inTables 4-8 is tested with one or more SNPs having the same Linkage Blockas listed in Tables 1A-C and 2 A-B for that same racial group.

IV. Methods of SNP Detection

Detection techniques for evaluating nucleic acids for the presence of aSNP involve procedures well known in the field of molecular genetics.Further, many of the methods involve amplification of nucleic acids.Ample guidance for performing is provided in the art. Exemplaryreferences include manuals such as PCR Technology: Principles andApplications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds.Innis, et al., Academic Press, San Diego, Calif., 1990); CurrentProtocols in Molecular Biology, Ausubel, 1994-1999, includingsupplemental updates through April 2004; Sambrook & Russell, MolecularCloning, A Laboratory Manual (3rd Ed, 2001).

Although the methods typically employ PCR steps, other amplification ornon-amplification-based protocols may also be used. Suitableamplification methods include ligase chain reaction (see, e.g., Wu &Wallace, Genomics 4:560-569, 1988); strand displacement assay (see,e.g., Walker et al., Proc. Natl. Acad. Sci. USA 89:392-396, 1992; U.S.Pat. No. 5,455,166); and several transcription-based amplificationsystems, including the methods described in U.S. Pat. Nos. 5,437,990;5,409,818; and 5,399,491; the transcription amplification system (TAS)(Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173-1177, 1989); andself-sustained sequence replication (3SR) (Guatelli et al., Proc. Natl.Acad. Sci. USA 87:1874-1878, 1990; WO 92/08800). Alternatively, methodsthat amplify the probe to detectable levels can be used, such asQβ-replicase amplification (Kramer & Lizardi, Nature 339:401-402, 1989;Lomeli et al., Clin. Chem. 35:1826-1831, 1989). A review of knownamplification methods is provided, for example, by Abramson and Myers inCurrent Opinion in Biotechnology 4:41-47, 1993.

Typically, detecting SNPs in an individual is performed usingoligonucleotide primers and/or probes. Oligonucleotides can be preparedby any suitable method, usually chemical synthesis. Oligonucleotides canbe synthesized using commercially available reagents and instruments.Alternatively, they can be purchased through commercial sources. Methodsof synthesizing oligonucleotides are well known in the art (see, e.g,Narang et al., Meth. Enzymol. 68:90-99, 1979; Brown et al., Meth.Enzymol. 68:109-151, 1979; Beaucage et al., Tetrahedron Lett.22:1859-1862, 1981; and the solid support method of U.S. Pat. No.4,458,066). In addition, modifications to the above-described methods ofsynthesis may be used to desirably impact enzyme behavior with respectto the synthesized oligonucleotides. For example, incorporation ofmodified phosphodiester linkages (e.g., phosphorothioate,methylphosphonates, phosphoamidate, or boranophosphate) or linkagesother than a phosphorous acid derivative into an oligonucleotide may beused to prevent cleavage at a selected site. In addition, the use of2′-amino modified sugars tends to favor displacement over digestion ofthe oligonucleotide when hybridized to a nucleic acid that is also thetemplate for synthesis of a new nucleic acid strand.

The amount and/or presence of an allele of a SNP of the invention in asample from an individual can be determined using many detection methodsthat are well known in the art. A number of SNP assay formats entail oneof several general protocols: hybridization using allele-specificoligonucleotides, primer extension, allele-specific ligation,sequencing, or electrophoretic separation techniques, e.g.,singled-stranded conformational polymorphism (SSCP) and heteroduplexanalysis. Exemplary assays include 5′ nuclease assays, template-directeddye-terminator incorporation, molecular beacon allele-specificoligonucleotide assays, single-base extension assays, and SNP scoring byreal-time pyrophosphate sequences. Analysis of amplified sequences canbe performed using various technologies such as microchips, fluorescencepolarization assays, and matrix-assisted laser desorption ionization(MALDI) mass spectrometry. Two methods that can also be used are assaysbased on invasive cleavage with Flap nucleases and methodologiesemploying padlock probes.

Determining the presence or absence of a particular SNP allele isgenerally performed by analyzing a nucleic acid sample that is obtainedfrom a biological sample from the individual to be analyzed. While theamount and/or presence of a SNP allele can be directly measured usingRNA from the sample, often times the RNA in a sample will be reversetranscribed, optionally amplified, and then the SNP allele will bedetected in the resulting cDNA.

Frequently used methodologies for analysis of nucleic acid samples tomeasure the amount and/or presence of an allele of a SNP are brieflydescribed. However, any method known in the art can be used in theinvention to measure the amount and/or presence of single nucleotidepolymorphisms.

Allele Specific Hybridization

This technique, also commonly referred to as allele specificoligonucleotide hybridization (ASO) (e.g., Stoneking et al., Am. J. Hum.Genet. 48:70-382, 1991; Saiki et al., Nature 324, 163-166, 1986; EP235,726; and WO 89/11548), relies on distinguishing between two DNAmolecules differing by one base by hybridizing an oligonucleotide probethat is specific for one of the variants to an amplified productobtained from amplifying the nucleic acid sample. In some embodiments,this method employs short oligonucleotides, e.g., 15-20 bases in length.The probes are designed to differentially hybridize to one variantversus another. Principles and guidance for designing such probe isavailable in the art, e.g., in the references cited herein.Hybridization conditions should be sufficiently stringent that there isa significant difference in hybridization intensity between alleles, andpreferably an essentially binary response, whereby a probe hybridizes toonly one of the alleles. Some probes are designed to hybridize to asegment of target DNA or cDNA such that the polymorphic site aligns witha central position (e.g., within 4 bases of the center of theoligonucleotide, for example, in a 15-base oligonucleotide at the 7position; in a 16-based oligonucleotide at either the 8 or 9 position)of the probe (e.g., a polynucleotide of the invention distinguishesbetween two SNP alleles as set forth herein), but this design is notrequired.

The amount and/or presence of an allele is determined by measuring theamount of allele-specific oligonucleotide that is hybridized to thesample. Typically, the oligonucleotide is labeled with a label such as afluorescent label. For example, an allele-specific oligonucleotide isapplied to immobilized oligonucleotides representing potential SNPsequences. After stringent hybridization and washing conditions,fluorescence intensity is measured for each SNP oligonucleotide.

In one embodiment, the nucleotide present at the polymorphic site isidentified by hybridization under sequence-specific hybridizationconditions with an oligonucleotide probe exactly complementary to one ofthe polymorphic alleles in a region encompassing the polymorphic site.The probe hybridizing sequence and sequence-specific hybridizationconditions are selected such that a single mismatch at the polymorphicsite destabilizes the hybridization duplex sufficiently so that it iseffectively not formed. Thus, under sequence-specific hybridizationconditions, stable duplexes will form only between the probe and theexactly complementary allelic sequence. Thus, oligonucleotides fromabout 10 to about 35 nucleotides in length, e.g., from about 15 to about35 nucleotides in length, which are exactly complementary to an allelesequence in a region which encompasses the polymorphic site are withinthe scope of the invention (e.g., one of SEQ ID NOs: 1-112).

In an alternative embodiment, the amount and/or presence of thenucleotide at the polymorphic site is identified by hybridization undersufficiently stringent hybridization conditions with an oligonucleotidesubstantially complementary to one of the SNP alleles in a regionencompassing the polymorphic site, and exactly complementary to theallele at the polymorphic site. Because mismatches that occur atnon-polymorphic sites are mismatches with both allele sequences, thedifference in the number of mismatches in a duplex formed with thetarget allele sequence and in a duplex formed with the correspondingnon-target allele sequence is the same as when an oligonucleotideexactly complementary to the target allele sequence is used. In thisembodiment, the hybridization conditions are relaxed sufficiently toallow the formation of stable duplexes with the target sequence, whilemaintaining sufficient stringency to preclude the formation of stableduplexes with non-target sequences. Under such sufficiently stringenthybridization conditions, stable duplexes will form only between theprobe and the target allele. Thus, oligonucleotides from about 10 toabout 35 nucleotides in length, preferably from about 15 to about 35nucleotides in length, which are substantially complementary to anallele sequence in a region which encompasses the polymorphic site, andare exactly complementary to the allele sequence at the polymorphicsite, are within the scope of the invention.

The use of substantially, rather than exactly, complementaryoligonucleotides may be desirable in assay formats in which optimizationof hybridization conditions is limited. For example, in a typicalmulti-target immobilized-probe assay format, probes for each target areimmobilized on a single solid support. Hybridizations are carried outsimultaneously by contacting the solid support with a solutioncontaining target DNA or cDNA. As all hybridizations are carried outunder identical conditions, the hybridization conditions cannot beseparately optimized for each probe. The incorporation of mismatchesinto a probe can be used to adjust duplex stability when the assayformat precludes adjusting the hybridization conditions. The effect of aparticular introduced mismatch on duplex stability is well known, andthe duplex stability can be routinely both estimated and empiricallydetermined, as described above. Suitable hybridization conditions, whichdepend on the exact size and sequence of the probe, can be selectedempirically using the guidance provided herein and well known in theart. The use of oligonucleotide probes to detect single base pairdifferences in sequence is described in, for example, Conner et al.,1983, Proc. Natl. Acad. Sci. USA 80:278-282, and U.S. Pat. Nos.5,468,613 and 5,604,099, each incorporated herein by reference.

The proportional change in stability between a perfectly matched and asingle-base mismatched hybridization duplex depends on the length of thehybridized oligonucleotides. Duplexes formed with shorter probesequences are destabilized proportionally more by the presence of amismatch. In practice, oligonucleotides between about 15 and about 35nucleotides in length are preferred for sequence-specific detection.Furthermore, because the ends of a hybridized oligonucleotide undergocontinuous random dissociation and re-annealing due to thermal energy, amismatch at either end destabilizes the hybridization duplex less than amismatch occurring internally. Preferably, for discrimination of asingle base pair change in target sequence, the probe sequence isselected which hybridizes to the target sequence such that thepolymorphic site occurs in the interior region of the probe.

The above criteria for selecting a probe sequence that hybridizes to aparticular SNP apply to the hybridizing region of the probe, i.e., thatpart of the probe which is involved in hybridization with the targetsequence. A probe may be bound to an additional nucleic acid sequence,such as a poly-T tail used to immobilize the probe, withoutsignificantly altering the hybridization characteristics of the probe.One of skill in the art will recognize that for use in the presentmethods, a probe bound to an additional nucleic acid sequence which isnot complementary to the target sequence and, thus, is not involved inthe hybridization, is essentially equivalent to the unbound probe.

Suitable assay formats for detecting hybrids formed between probes andtarget nucleic acid sequences in a sample are known in the art andinclude the immobilized target (dot-blot) format and immobilized probe(reverse dot-blot or line-blot) assay formats. Dot blot and reverse dotblot assay formats are described in U.S. Pat. Nos. 5,310,893; 5,451,512;5,468,613; and 5,604,099; each incorporated herein by reference.

In a dot-blot format, amplified target DNA or cDNA is immobilized on asolid support, such as a nylon membrane. The membrane-target complex isincubated with labeled probe under suitable hybridization conditions,unhybridized probe is removed by washing under suitably stringentconditions, and the membrane is monitored for the presence of boundprobe.

In the reverse dot-blot (or line-blot) format, the probes areimmobilized on a solid support, such as a nylon membrane or a microtiterplate. The target DNA or cDNA is labeled, typically during amplificationby the incorporation of labeled primers. One or both of the primers canbe labeled. The membrane-probe complex is incubated with the labeledamplified target DNA or cDNA under suitable hybridization conditions,unhybridized target DNA or cDNA is removed by washing under suitablystringent conditions, and the membrane is monitored for the presence ofbound target DNA or cDNA.

An allele-specific probe that is specific for one of the polymorphismvariants is often used in conjunction with the allele-specific probe forthe other polymorphism variant. In some embodiments, the probes areimmobilized on a solid support and the target sequence in an individualis analyzed using both probes simultaneously. Examples of nucleic acidarrays are described by WO 95/11995. The same array or a different arraycan be used for analysis of characterized polymorphisms. WO 95/11995also describes subarrays that are optimized for detection of variantforms of a pre-characterized polymorphism.

Allele-Specific Primers

The amount and/or presence of an allele is also commonly detected usingallele-specific amplification or primer extension methods. Thesereactions typically involve use of primers that are designed tospecifically target a polymorphism via a mismatch at the 3′ end of aprimer. The presence of a mismatch affects the ability of a polymeraseto extend a primer when the polymerase lacks error-correcting activity.For example, to detect an allele sequence using an allele-specificamplification- or extension-based method, a primer complementary to thepolymorphic nucleotide of a SNP is designed such that the 3′ terminalnucleotide hybridizes at the polymorphic position. The presence of theparticular allele can be determined by the ability of the primer toinitiate extension. If the 3′ terminus is mismatched, the extension isimpeded. If a primer matches the polymorphic nucleotide at the 3′ end,the primer will be efficiently extended.

Typically, the primer is used in conjunction with a second primer in anamplification reaction. The second primer hybridizes at a site unrelatedto the polymorphic position. Amplification proceeds from the two primersleading to a detectable product signifying the particular allelic formis present. Allele-specific amplification- or extension-based methodsare described in, for example, WO 93/22456; U.S. Pat. Nos. 5,137,806;5,595,890; 5,639,611; and U.S. Pat. No. 4,851,331.

Using allele-specific amplification-based methods, identification and/orquantification of the alleles require detection of the presence orabsence of amplified target sequences. Methods for the detection ofamplified target sequences are well known in the art. For example, gelelectrophoresis and probe hybridization assays described are often usedto detect the presence of nucleic acids.

In an alternative probe-less method, the amplified nucleic acid isdetected by monitoring the increase in the total amount ofdouble-stranded DNA in the reaction mixture, is described, e.g., in U.S.Pat. No. 5,994,056; and European Patent Publication Nos. 487,218 and512,334. The detection of double-stranded target DNA or cDNA relies onthe increased fluorescence various DNA-binding dyes, e.g., SYBR Green,exhibit when bound to double-stranded DNA.

As appreciated by one in the art, allele-specific amplification methodscan be performed in reactions that employ multiple allele-specificprimers to target particular alleles. Primers for such multiplexapplications are generally labeled with distinguishable labels or areselected such that the amplification products produced from the allelesare distinguishable by size. Thus, for example, both alleles in a singlesample can be identified and/or quantified using a single amplificationby various methods.

As in the case of allele-specific probes, an allele-specificoligonucleotide primer may be exactly complementary to one of thepolymorphic alleles in the hybridizing region or may have somemismatches at positions other than the 3′ terminus of theoligonucleotide, which mismatches occur at non-polymorphic sites in bothallele sequences.

5′-Nuclease Assay

The amount and/or presence of an allele can also be determined using a“TaqMan®” or “5′-nuclease assay”, as described in U.S. Pat. Nos.5,210,015; 5,487,972; and 5,804,375; and Holland et al., 1988, Proc.Natl. Acad. Sci. USA 88:7276-7280. In the TaqMan® assay, labeleddetection probes that hybridize within the amplified region are addedduring the amplification reaction. The probes are modified so as toprevent the probes from acting as primers for DNA synthesis. Theamplification is performed using a DNA polymerase having 5′ to 3′exonuclease activity. During each synthesis step of the amplification,any probe which hybridizes to the target nucleic acid downstream fromthe primer being extended is degraded by the 5′ to 3′ exonucleaseactivity of the DNA polymerase. Thus, the synthesis of a new targetstrand also results in the degradation of a probe, and the accumulationof degradation product provides a measure of the synthesis of targetsequences.

The hybridization probe can be an allele-specific probe thatdiscriminates between the SNP alleles. Alternatively, the method can beperformed using an allele-specific primer and a labeled probe that bindsto amplified product.

Any method suitable for detecting degradation product can be used in a5′ nuclease assay. Often, the detection probe is labeled with twofluorescent dyes, one of which is capable of quenching the fluorescenceof the other dye. The dyes are attached to the probe, preferably oneattached to the 5′ terminus and the other is attached to an internalsite, such that quenching occurs when the probe is in an unhybridizedstate and such that cleavage of the probe by the 5′ to 3′ exonucleaseactivity of the DNA polymerase occurs in between the two dyes.Amplification results in cleavage of the probe between the dyes with aconcomitant elimination of quenching and an increase in the fluorescenceobservable from the initially quenched dye. The accumulation ofdegradation product is monitored by measuring the increase in reactionfluorescence. U.S. Pat. Nos. 5,491,063 and 5,571,673, both incorporatedherein by reference, describe alternative methods for detecting thedegradation of probe which occurs concomitant with amplification.

DNA Sequencing and Single Base or Other Primer Extensions

The amount and/or presence of an allele can also be determined by directsequencing. Methods include e.g., dideoxy sequencing-based methods andother methods such as Maxam and Gilbert sequence (see, e.g., Sambrookand Russell, supra).

Other detection methods include Pyrosequencing™ ofoligonucleotide-length products. Such methods often employ amplificationtechniques such as PCR. For example, in pyrosequencing, a sequencingprimer is hybridized to a single stranded, PCR-amplified, DNA or cDNAtemplate; and incubated with the enzymes, DNA polymerase, ATPsulfurylase, luciferase and apyrase, and the substrates, adenosine 5′phosphosulfate (APS) and luciferin. The first of four deoxynucleotidetriphosphates (dNTP) is added to the reaction. DNA polymerase catalyzesthe incorporation of the deoxynucleotide triphosphate into the DNAstrand, if it is complementary to the base in the template strand. Eachincorporation event is accompanied by release of pyrophosphate (PPi) ina quantity equimolar to the amount of incorporated nucleotide. ATPsulfurylase quantitatively converts PPi to ATP in the presence ofadenosine 5′ phosphosulfate. This ATP drives the luciferase-mediatedconversion of luciferin to oxyluciferin that generates visible light inamounts that are proportional to the amount of ATP. The light producedin the luciferase-catalyzed reaction is detected by a charge coupleddevice (CCD) camera and seen as a peak in a Pyrogram™. Each light signalis proportional to the number of nucleotides incorporated. Apyrase, anucleotide degrading enzyme, continuously degrades unincorporated dNTPsand excess ATP. When degradation is complete, another dNTP is added.

Another similar method for characterizing SNPs does not require use of acomplete PCR, but typically uses only the extension of a primer by asingle, detectably (e.g., fluorescently)-labeled dideoxyribonucleic acidmolecule (ddNTP) that is complementary to the nucleotide to beinvestigated. The nucleotide at the polymorphic site can be identifiedvia detection of a primer that has been extended by one base and isfluorescently labeled (e.g., Kobayashi et al, Mol. Cell. Probes,9:175-182, 1995). Of course extension products can also be detectedbased on other types of labels, or by mass-spectrometry, as desired.

In a similar method, PCR amplified target DNA or RT-PCR amplified targetcDNA may be used as template for a single nucleotide primer extensionreaction whereby a single fluorescently labeled ddNTP complementary tothe polymorphic nucleotide is incorporated on the 3′ end of a singleprimer. Each specific ddNTP can be labeled with a different fluorescentdye (eg. ddATP labeled with dR6G, ddCTP labeled with dTAMRA™, ddGTPlabeled with dR110 and ddTTP or ddUTP labeled with dROX™). Therefore,single nucleotide extension of the initially unlabeled primer tags theprimer with a specific fluorescent dye that identifies the base that wasadded to the 3′ end of the unlabeled primer. Extended primers can beresolved and analyzed to determine the presence and relative quantity ofeach specific dye-tagged primer, representing the relative quantities ofeach allele in the target DNA or target cDNA template.

Restriction Fragment Length Polymorphism Analysis

In other embodiments, the amount and/or presence of an allele of a SNPcan be determined by differential digestion of amplified target DNA orcDNA when the polymorphic nucleotide of interest lies within therecognition sequence of a restriction enzyme. In one case, one allele ofthe SNP (the first allele) maintains the recognition sequence of therestriction enzyme and the other allele (the second allele) does not. Inthis case, the restriction enzyme will cleave the target DNA or cDNAincluding the first allele, but not the target DNA or cDNA including thesecond allele. In another case, one allele (the first allele) of the SNPmaintains the recognition sequence of a restriction enzyme (the firstrestriction enzyme) and the other allele (the second allele) maintainsthe recognition sequence of a different restriction enzyme (the secondrestriction enzyme). In this case, the first restriction enzyme willcleave the target DNA or cDNA including the first allele, but not thetarget DNA or cDNA including the second allele. The second restrictionenzyme will cleave the target DNA or cDNA including the second allele,but not the target DNA of cDNA including the first allele. The amountand/or presence of alleles can be determined by various methodsincluding, but not limited to, Southern blot hybridization toimmobilized restricted fragments and quantification of band intensities,resolution and visualization of restriction fragments by gelelectrophoresis, resolution and quantification of restriction fragmentsby capillary electrophoresis (such as performed using an AgilentBioAnalyzer), or differential quantitative PCR amplification of cleavedversus uncleaved template DNA or cDNA.

Denaturing Gradient Gel Electrophoresis

Amplification products generated using the polymerase chain reaction canbe analyzed by the use of denaturing gradient gel electrophoresis.Different alleles can be identified based on the differentsequence-dependent melting properties and electrophoretic migration ofDNA in solution (see, e.g., Erlich, ed., PCR Technology, Principles andApplications for DNA Amplification, W. H. Freeman and Co, New York,1992, Chapter 7).

Single-Strand Conformation Polymorphism Analysis

Alleles of target sequences can be differentiated using single-strandconformation polymorphism analysis, which identifies base differences byalteration in electrophoretic migration of single stranded PCR products,as described, e.g, in Orita et al., Proc. Nat. Acad. Sci. 86, 2766-2770(1989). Amplified PCR or RT-PCR products can be generated as describedabove, and heated or otherwise denatured, to form single strandedamplification products. Single-stranded nucleic acids may refold or formsecondary structures which are partially dependent on the base sequence.The different electrophoretic mobilities of single-strandedamplification products can be related to base-sequence differencebetween alleles of target

SNP detection methods often employ labeled oligonucleotides.Oligonucleotides can be labeled by incorporating a label detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. Useful labels include fluorescent dyes, radioactive labels, e.g.,³²P, electron-dense reagents, enzyme, such as peroxidase or alkalinephosphatase, biotin, or haptens and proteins for which antisera ormonoclonal antibodies are available. Labeling techniques are well knownin the art (see, e.g., Current Protocols in Molecular Biology, supra;Sambrook & Russell, supra).

V. Methods for Quantifying RNA

The presence and quantity of RNA corresponding to a particular SNP canbe readily determined according to any method for quantifying RNA.Various methods involving linkage of RNA to a solid support and probingthe RNA (e.g., northern blots, dot blots, etc.) can be used.

In some embodiments, the target RNA is first reverse transcribed (e.g.,with reverse transcriptase) and then the resulting cDNA is quantified byany methods known in the art (blot hybridization, RT-PCR, etc.) as asurrogate for RNA quantity. Various methods of reverse transcription areknown and described, e.g., in Sambrook et al., Molecular Cloning, ALaboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994)), and can involve reversetranscription using either specific or non-specific primers.

In some embodiments, RT-PCR or other quantitative amplificationtechniques are used to quantify the target RNA. Amplification of cDNAusing reactions is well known (see U.S. Pat. Nos. 4,683,195 and4,683,202; PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS (Innis etal., eds, 1990)).

Sequences amplified by the methods of the invention can be furtherevaluated, detected, cloned, sequenced, and the like, either in solutionor after binding to a solid support, by any method usually applied tothe detection of a specific DNA sequence such as PCR, oligomerrestriction (Saiki, et al., Bio/Technology 3:1008-1012 (1985)),allele-specific oligonucleotide (ASO) probe analysis (Conner, et al.,PNAS USA 80:278 (1983)), oligonucleotide ligation assays (OLAs)(Landegren, et al., Science 241:1077, (1988)), and the like. Moleculartechniques for DNA analysis have been reviewed (Landegren, et al.,Science 242:229-237 (1988)).

Methods of quantitative amplification are disclosed in, e.g., U.S. Pat.Nos. 6,180,349; 6,033,854; and 5,972,602, as well as in, e.g., Gibson etal., Genome Research 6:995-1001 (1996); DeGraves, et al., Biotechniques34(1):106-10, 112-5 (2003); Deiman B, et al., Mol Biotechnol.20(2):163-79 (2002). Amplifications may be monitored in “real time.”

In general, quantitative amplification is based on the monitoring of thesignal (e.g., fluorescence of a probe) representing copies of thetemplate in cycles of an amplification (e.g., PCR) reaction. In theinitial cycles of the PCR, a very low signal is observed because thequantity of the amplicon formed does not support a measurable signaloutput from the assay. After the initial cycles, as the amount of formedamplicon increases, the signal intensity increases to a measurable leveland reaches a plateau in later cycles when the PCR enters into anon-logarithmic phase. Through a plot of the signal intensity versus thecycle number, the specific cycle at which a measurable signal isobtained from the PCR reaction can be deduced and used to back-calculatethe quantity of the target before the start of the PCR. The number ofthe specific cycles that is determined by this method is typicallyreferred to as the cycle threshold (Ct). Exemplary methods are describedin, e.g., Heid et al. Genome Methods 6:986-94 (1996) with reference tohydrolysis probes.

One method for detection of amplification products is the 5′-3′exonuclease “hydrolysis” PCR assay (also referred to as the TaqMan™assay) (U.S. Pat. Nos. 5,210,015 and 5,487,972; Holland et al., PNAS USA88: 7276-7280 (1991); Lee et al., Nucleic Acids Res. 21: 3761-3766(1993)). This assay detects the accumulation of a specific PCR productby hybridization and cleavage of a doubly labeled fluorogenic probe (the“TaqMan™” probe) during the amplification reaction. The fluorogenicprobe consists of an oligonucleotide labeled with both a fluorescentreporter dye and a quencher dye. During PCR, this probe is cleaved bythe 5′-exonuclease activity of DNA polymerase if, and only if, ithybridizes to the segment being amplified. Cleavage of the probegenerates an increase in the fluorescence intensity of the reporter dye.

Another method of detecting amplification products that relies on theuse of energy transfer is the “beacon probe” method described by Tyagiand Kramer, Nature Biotech. 14:303-309 (1996), which is also the subjectof U.S. Pat. Nos. 5,119,801 and 5,312,728. This method employsoligonucleotide hybridization probes that can form hairpin structures.On one end of the hybridization probe (either the 5′ or 3′ end), thereis a donor fluorophore, and on the other end, an acceptor moiety. In thecase of the Tyagi and Kramer method, this acceptor moiety is a quencher,that is, the acceptor absorbs energy released by the donor, but thendoes not itself fluoresce. Thus, when the beacon is in the openconformation, the fluorescence of the donor fluorophore is detectable,whereas when the beacon is in hairpin (closed) conformation, thefluorescence of the donor fluorophore is quenched. When employed in PCR,the molecular beacon probe, which hybridizes to one of the strands ofthe PCR product, is in “open conformation,” and fluorescence isdetected, while those that remain unhybridized will not fluoresce (Tyagiand Kramer, Nature Biotechnol. 14: 303-306 (1996)). As a result, theamount of fluorescence will increase as the amount of PCR productincreases, and thus may be used as a measure of the progress of the PCR.Those of skill in the art will recognize that other methods ofquantitative amplification are also available.

Various other techniques for performing quantitative amplification of anucleic acids are also known. For example, some methodologies employ oneor more probe oligonucleotides that are structured such that a change influorescence is generated when the oligonucleotide(s) is hybridized to atarget nucleic acid. For example, one such method involves is a dualfluorophore approach that exploits fluorescence resonance energytransfer (FRET), e.g., LightCycler™ hybridization probes, where twooligo probes anneal to the amplicon. The oligonucleotides are designedto hybridize in a head-to-tail orientation with the fluorophoresseparated at a distance that is compatible with efficient energytransfer. Other examples of labeled oligonucleotides that are structuredto emit a signal when bound to a nucleic acid or incorporated into anextension product include: Scorpions™ probes (e.g., Whitcombe et al.,Nature Biotechnology 17:804-807, 1999, and U.S. Pat. No. 6,326,145),Sunrise™ (or Amplifluor™) probes (e.g., Nazarenko et al., Nuc. AcidsRes. 25:2516-2521, 1997, and U.S. Pat. No. 6,117,635), and probes thatform a secondary structure that results in reduced signal without aquencher and that emits increased signal when hybridized to a target(e.g., Lux Probes™)□.

In other embodiments, intercalating agents that produce a signal whenintercalated in double stranded DNA may be used. Exemplary agentsinclude SYBR GREEN™ and SYBR GOLD™. Since these agents are nottemplate-specific, it is assumed that the signal is generated based ontemplate-specific amplification. This can be confirmed by monitoringsignal as a function of temperature because melting point of templatesequences will generally be much higher than, for example,primer-dimers, etc.

VI. Kits

The invention also provides kits comprising useful components forpracticing the methods. In some embodiments, the kit may comprise one orboth allele-specific detection polynucleotides (e.g., primers or probes)for a SNP of the invention, which optionally can be fixed to anappropriate support membrane. In some embodiments, the kits comprise afirst isolated polynucleotide of between 8-100 nucleotides, wherein thepolynucleotide distinguishes between a first allele of a SNP (or acomplement thereof) and a second allele of the SNP (or a complementthereof) in a hybridization reaction, and optionally a second isolatedpolynucleotide of between 8-100 nucleotides, wherein the polynucleotidedistinguishes between the first allele of the SNP (or a complementthereof) and the second allele of the SNP (or a complement thereof), andwherein the first polynucleotide is complementary to the polymorphicnucleotide in the first allele and the second polynucleotide iscomplementary to the polymorphic nucleotide of the second allele.Optionally, the kits comprise one or both allele specificpolynucleotides for 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or moreSNPs selected from SEQ ID NOs: 1-112 Such a kit can also containamplification primers for amplifying a region of the IGF2 locusencompassing the polymorphic site. Alternatively, useful kits cancontain a set of primers comprising an allele-specific primer for thespecific amplification of the polymorphic alleles. Such a kit may alsocomprises probes for the detection of amplification products.Alternatively, useful kits can contain a set of primers complementary tosequences 5′ to but not including the SNP positions of interest (orcomplements thereof) for use in primer extension methods as describedabove.

Other optional components of the kits include additional reagents usedfor genotyping patients and/or quantifying the relative amount ofspecific alleles present. For example, a kit can contain a polymerase,labeled or unlabeled substrate nucleoside triphosphates, means forlabeling and/or detecting nucleic acid, appropriate buffers foramplification or hybridization reactions, and instructions for carryingout the present method.

VII. Reaction Mixtures

The invention also provides reaction mixtures comprising components forpracticing the methods. In some embodiments, the kit may comprise one orboth allele-specific detection polynucleotides (e.g., primers or probes)for a SNP (or a complement thereof) of the invention, which optionallycan be fixed to an appropriate support membrane. In some embodiments,the reaction mixtures comprise a first isolated polynucleotide ofbetween 8-100 nucleotides, wherein the polynucleotide distinguishesbetween a first allele of a SNP (or a complement thereof) and a secondallele of the SNP (or a complement thereof) in a hybridization reaction,and optionally a second isolated polynucleotide of between 8-100nucleotides, wherein the polynucleotide distinguishes between the firstallele of the SNP (or a complement thereof) and the second allele of theSNP (or a complement thereof), and wherein the first polynucleotide iscomplementary to the polymorphic nucleotide in the first allele and thesecond polynucleotide is complementary to the polymorphic nucleotide ofthe second allele. Optionally, the reaction mixtures comprise one orboth allele specific polynucleotides for 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,50, 100, or more SNPs selected from SEQ ID NO: 1-112. Such reactionmixtures can also contain amplification primers for amplifying a regionof the IGF2 locus encompassing the polymorphic site. Alternatively,reaction mixtures can contain a set of primers comprising anallele-specific primer for the specific amplification of the polymorphicalleles. Such a reaction mixture may also comprise probes for thedetection of amplification products. Optionally, reaction mixturescomprise a set of primers complementary to sequences 5′ to but notincluding the SNP positions for 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100,or more SNPs selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, and 112.

Other optional components of the reaction mixtures include additionalreagents used for genotyping patients and/or quantifying the relativeamount of specific alleles present. For example, a reaction mixture cancontain a polymerase, labeled or unlabeled substrate nucleosidetriphosphates, means for labeling and/or detecting nucleic acid,appropriate buffers for amplification or hybridization reactions.

VIII. Cancer Detection

IGF2 LOI is associated with, for example, a predisposition of cancer aswell as predicting the efficacy of treatment of cancer using variousdrugs. See, e.g., WO2004/003003; Kaneda et al. Proc. Natl. Acad. Sci.USA 104(52):20926-20931 (2007). Accordingly, detection of LOI in IGF2 asdescribed herein can be used in the diagnosis, prognosis,classification, prediction of cancer risk, detection of recurrence ofcancer, and selection of treatment of a number of types of cancers. Acancer at any stage of progression can be detected, such as primary,metastatic, and recurrent cancers. Information regarding numerous typesof cancer can be found, e.g., from the American Cancer Society(available on the worldwide web at cancer.org), or from, e.g.,Harrison's Principles of Internal Medicine, Kaspar, et al., eds., 16thEdition, 2005, McGraw-Hill, Inc. Exemplary cancers that can be detectedinclude bladder, breast, cervical, choriocarcinoma, colorectal neoplasia(colorectal adenoma or colorectal cancer), esophageal, gastricadenocarcinoma, glioma, hepatocellular, acute myeloid leukemia, chronicmyelogenous leukemia, lung, medulloblastoma, prostate, mesothelioma,ovarian, renal cell carcinoma, testicular germ cell, and uterine cancer.

The present invention provides methods for determining whether or not amammal (e.g., a human) has cancer, whether or not a biological sampletaken from a mammal contains cancerous cells, estimating the risk orlikelihood of a mammal developing cancer, classifying cancer types andstages, monitoring the efficacy of anti-cancer treatment, or selectingthe appropriate anti-cancer treatment in a mammal with cancer.

In some embodiments, the biological sample comprises a tissue samplefrom a tissue suspected of containing cancerous cells. For example, inan individual suspected of having cancer, breast tissue, lymph tissue,lung tissue, brain tissue, or blood can be evaluated. Alternatively,lung, renal, liver, ovarian, head and neck, thyroid, bladder, cervical,colon, endometrial, esophageal, prostate, or skin tissue can beevaluated. The tissue or cells can be obtained by any method known inthe art including, e.g., by surgery, biopsy, phlebotomy, swab, nippledischarge, stool, etc. In other embodiments, a tissue sample known tocontain cancerous cells, e.g., from a tumor, will be analyzed for thepresence or quantity of methylation at one or more of the diagnosticbiomarkers of the invention to determine information about the cancer,e.g., the efficacy of certain treatments, the survival expectancy of theindividual, etc. In some embodiments, the methods will be used inconjunction with additional diagnostic methods, e.g., detection of othercancer biomarkers, etc.

The methods of the invention can be used to evaluate individuals knownor suspected to have cancer or as a routine clinical test, i.e., in anindividual not necessarily suspected to have cancer. Further diagnosticassays can be performed to confirm the status of cancer in theindividual.

Further, the present methods may be used to assess the efficacy of acourse of treatment. For example, the efficacy of an anti-cancertreatment can be assessed by monitoring LOI over time in a mammal havingcancer. For example, a reduction or absence of LOI in IGF2 in abiological sample taken from a mammal following a treatment, compared toa level in a sample taken from the mammal before, or earlier in, thetreatment, indicates efficacious treatment. Further, a patient can bescreened for LOI of IGF2 prior to selection of an appropriate drug forcancer treatment. For example, once LOI is detected, the patient islikely a good candidate for an IGF1R inhibitor. See, e.g., Kaneda etal., supra.

In some embodiments, the methods comprise recording a diagnosis,prognosis, risk assessment or classification, based on the methylationstatus determined from an individual. Any type of recordation iscontemplated, including electronic recordation, e.g., by a computer.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Discovery of Novel SNPs within Exon 9 of the IGF2 Gene

A collection of SNPs within exon 9 of the IGF2 gene have previously beenreported. Tables 1A, 1B and 1C list the genomic coordinates (NCBI build36), single nucleotide sequence variants, NCBI dbSNP referenceidentifier and surrounding nucleotide sequences of previously identifiedSNPs (dbSNP build 129). To identify previously uncharacterized SNPs, wedesigned 15 PCR amplicons that tile the majority of IGF2 exon 9. Eachwas used to amplify by PCR the genomic DNA derived from a panel of 589individuals, including 462 samples that are part of the InternationalHapMap Project collection. The panel included 225 Caucasian individuals(98 from Coriell Human Variation Panel including unrelated healthyCaucasian individuals and 127 individuals from which blood samples werecommercially obtained), 96 African American individuals (Coriell HumanVariation Panel), 96 individuals of Mexican descent (Coriell HumanVariation panel of Mexican-American Community of Los Angeles includingunrelated individuals, each having either three or four grandparentsborn in Mexico), 88 Japanese individuals (International Hapmap Projectcollection including individuals from Tokyo, Japan), and 84 Chineseindividuals (International Hapmap Project collection including HanChinese individuals from Beijing, China). Multiple direct sequencingattempts were made in both directions for all amplicons. Sequences wereassembled and aligned, genotypes were scored, and SNPs were identifiedfor each person in the panel by an automated polyphred and polyscansequencing analysis pipeline. Genotype designations for highheterozygosity frequency SNPs were manually confirmed by manualinspection of sequence chromatograms within CONSED. As an additionalmeasure of the confidence of genotype designations based on sequencingdata, an independent restriction enzyme based genotyping assay wasdesigned for the SNP corresponding to SEQ ID NO: 64 in Table 1A, asdescribed in Example 4. Genotype designations were compared to thosebased on the sequencing data. Seventy individuals from the Caucasianpanel were genotyped by both methods. The concordance between genotypedesignations based on the two independent methods (sequencing andrestriction enzyme digestion based assays) was 100%.

Tables 2A and 2B list the genomic coordinates (NCBI build 36) of singlenucleotide sequence variants and surrounding nucleotide sequences ofnovel SNPs discovered in the study described in the present application.The observed frequencies of heterozygosity for selected SNPs (includingboth novel and previously identified SNPs) in the sequenced panels ofall individuals, as well as in the African American, Caucasian, Chinese,Japanese and Mexican individuals are listed in Table 3. Theidentification of novel SNPs implies that the novel SNPs described inthe present application can be useful for improved detection of LOI ofIGF2. For example, the observed heterozygosity frequency of SEQ ID 10among individuals in the African American panel is 17.33%.

This study demonstrates the differential heterozygosity frequencies ofboth novel and previously identified SNPs between different racialgroups. SNPs that were genotyped as heterozygous in at least 2% ofindividuals within the Chinese, Japanese, African American, Caucasian,and Mexican cohorts are listed in Tables 4-8, respectively. Therefore,the optimal SNP or combinations of SNPs for monitoring LOI of IGF2 canvary between racial groups.

Example 2 Use of any One of the Novel SNPs for Improved Detection of LOIof the IGF2 Gene

As described above, the detection of LOI of the IGF2 gene is based onthe independent comparison of the amount of expression derived from eachof the two copies of the IGF2 gene isolated from a biological samplefrom a given individual. The IGF2 gene is normally maternally imprinted,(i.e. the copy inherited from an individual's mother is normallytranscriptionally repressed), while the paternally inherited copy of thegene is normally expressed. LOI occurs when the IGF2 maternal imprint isrelaxed, resulting in similar expression levels of both the paternallyand maternally inherited copies of the gene. One method of measuring theimprinting status of IGF2 in a sample is to first isolate genomic DNAfrom a biological sample and then determine the genotype(s) of one ormore polymorphic sites in the transcribed region of the IGF2 gene.Second, allele-specific expression of IGF2 is then measured by utilizingone or more heterozygous nucleotides in RNA that is extracted from thesame biological sample. Expression from each of the two copies of theIGF2 gene may be independently measured with an assay(s) that isquantitative, and that can sufficiently discriminate between the twoalleles of one or more heterozygous SNPs within the sample. Third, aratio of the amount of expression from one allele to the amount ofexpression of the other allele is computed and compared to a thresholdvalue, thereby determining the imprinting status of the IGF2 gene in thesample.

As an example of the utility of any one or more of the novel SNPsreported in the present application for monitoring LOI of IGF2, onespecific intended approach is described here. It is apparent to thoseskilled in the art that multiple approaches for detection andquantification of SNPs exist, and any of these may be utilized for boththe genotyping of genomic DNA from a biological sample for a particularSNP and the quantification of relative levels of each sequence variantpresent in expressed mRNA of a biological sample. A basic strategy isoutlined in FIG. 2. This involves isolating both genomic DNA and totalor polyadenylated RNA from a biological sample (for example, peripheralblood, peripheral blood mononuclear cells, colonic mucosa sample, stoolsample, etc.) derived from an individual. The genomic DNA sample is thengenotyped with assays detecting the alleles of one or more SNP. Thisstep determines what SNPs, if any, may be utilized for analysis ofallele-specific expression of the IGF2 gene in the matched RNA sample.If the individual is homozygous for all SNPs evaluated by an assay, theindividual is not informative for those SNPs and can not be measured forLOI of IGF2. If the individual is heterozygous (informative) for one ormore SNPs, cDNA is amplified from the relevant region of the IGF2transcript using standard reverse transcriptase/PCR (RT-PCR) methods.Expression from each of the two copies of the IGF2 gene is independentlymeasured using the generated cDNA with an assay that is quantitative,and that can sufficiently discriminate between the two alleles.Computation of the ratio of the amount of expression of one allelerelative to the amount of expression of the other allele, and comparisonof this ratio to a threshold value determines the imprinting status ofthe IGF2 gene. If multiple heterozygous SNPs exist for a given sample,assays that discriminate each of the SNPs may be used simultaneously.This allows redundant measurements of allele-specific expression withina sample, and comparison of these measurements may be used to determinethe accuracy of the determination of LOI. While a range of thresholdvalues can be used, typically, a sample is classically determined todisplay LOI of IGF2 if the quantified proportion of the lesser abundantallele is greater than or equal to 33.3% the quantified proportion ofthe more abundant allele.

One method for genotyping an individual for a given SNP is accomplishedby designing an oligonucleotide primer that is complementary to thesequence of the IGF2 gene and that has a 3′ terminal nucleotide that iscomplementary to the IGF2 template nucleotide one base 3′ to thetemplate polymorphic nucleotide (see FIG. 7 for example). Assays may bedesigned to genotype any one or more of the SNPs listed in Tables 1A,1B, 1C, 2A and 2B. The oligonucleotide primer is combined with andhybridized to the PCR amplified DNA product from the genomic DNA samplein a mixture including all ddNTPs (ddATP, ddCTP, ddGTP, ddTTP (orddUTP)), each tagged with a different fluorescent moiety. For example,if a G/A polymorphism is to be genotyped (and the G/A nucleotide is onthe template strand of the genomic DNA sample), the oligonucleotideprimer is designed to hybridize to the complementary template with its3′ terminal nucleotide hybridized to the complementary templatenucleotide one base 3′ to the template G/A position. Single nucleotideprimer extension is catalyzed by a DNA polymerase in the presence of thedifferentially fluorescently labeled ddNTPs such that oligonucleotidesthat extend by incorporation of ddCTP (representing the G allele) or byincorporation of ddTTP (representing the A allele) are differentiallyfluorescently labeled at their 3′ termini. Extended oligonucleotides arethen resolved by capillary electrophoresis and analyzed in the presenceof a fifth-fluorescent dye-labeled size standard. Peaks representingspecific single nucleotide primer extension products are detected andquantified to determine the genotype for the given DNA sample. MultipleSNPs may be genotyped in one reaction by multiplexing witholigonucleotides of different lengths designed to terminate just 3′ todifferent polymorphic sites. Different genotypes are obtained based oni) resolution of different length extended oligonucleotides and ii) thespecific fluorescent tagged ddNTP incorporated during single nucleotideextension.

One method for determining the imprinting status of IGF2 involves ananalogous single nucleotide primer extension approach that is designedto discriminate different alleles of a particular SNP. Assays may bedesigned to utilize any one or more of the SNPs listed in Tables 1 and2. If a given SNP is determined to be heterozygous in a genomic DNAsample, first strand cDNA is amplified from the matched RNA sample by areverse transcriptase (RT) using random hexamer or decamer primers,oligodT primers complementary to polyA tails of mRNA or a primercomplementary to a specific region of the IGF2 transcript.Oligonucleotide primers complementary to sequences flanking the SNP siteare subsequently used to PCR amplify a cDNA product including thepolymorphic site. Alternatively, nested PCR approaches may be used togenerate cDNA products. Alternatively, approaches including generationof aRNA from cDNA by linear in vitro transcription, followed by a secondreverse transcription reaction using random hexomer or decamer primersor IGF2 transcript-specific primer and subsequent PCR amplification maybe used to generate cDNA products. These RT-PCR products are thenassayed for the specific sequence variants of the polymorphic site usingthe same single nucleotide primer extension assay(s) described above.Peaks representing specific single nucleotide primer extension productsare detected and quantified. The ratio of the quantified amount of oneallele to the other allele is determined. LOI is detected if thequantified proportion of the PCR product representing the lesserabundant allele is greater than or equal to 33.3% the quantifiedproportion of the PCR product representing the more abundant allele. Asdescribed above, multiple heterozygous SNPs may be used to measure LOIin a common reaction by multiplexing with oligonucleotides of differentlengths designed to terminate just 3′ to different polymorphic sites orwith oligonucleotides that incorporate different labeled ddNTPs into theextended primer.

Example 3 Linkage Analysis Indicates Minimal Linkage DisequilibriumAmong Numerous SNPs

The present application describes the discovery of numerous novel SNPsin exon 9 of IGF2. These data allowed high resolution haplotype analysesof 589 individuals (see example 1 for a description of the discoverypanel). Genotype data was analyzed by Haploview (Broad Institute of MITand Harvard University) to determine the presence, or absence, ofhaplotype blocks across the analyzed regions.

FIG. 4 shows haplotype analyses of SNPs distributed throughout the IGF2exon 9 region in the entire individual panel. SNPs included in theanalysis displayed at least 1% heterozygosity frequency within thegenotyped individuals.

Across all individuals (FIG. 4), eight SNPs distributed throughout exon9 were analyzed (SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 64, SEQ ID NO:10, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 32, SEQ ID NO: 102).Included in the eight SNP subset is SEQ ID 10, which, of all novel SNPsincluded in this study, had the highest heterozygosity frequency withina given cohort (17.33% heterozygosity frequency in the African Americancohort). This study demonstrated minimal linkage disequilibrium betweenSNPs within the previously reported haplotype block (i.e. within “GauntBlock 2” as indicated in FIG. 4). For example, strong evidence forhistorical recombination was detected between the high frequency SNP SEQID NO: 10 and SEQ ID NO: 16 (indicated by the white diamond in theHaploview output). Minimal linkage disequilibrium was also observedbetween SNPs within the previously reported haplotype block and SNPswithin the up and downstream regions of exon 9 (uncharacterized regionsblock 1 and block 3). One example of this is the high frequency SNP inblock 2, SEQ ID 10, which reported minimal linkage disequilibrium withSEQ ID 102 in block 3. Additional examples of these SNP pairs includeSEQ ID NO: 4 and SEQ ID NO: 64; SEQ ID NO: 4 and SEQ ID NO: 16; SEQ IDNO: 64 and SEQ ID NO: 32; and SEQ ID NO: 10 and SEQ ID NO: 102.Surprisingly, and contrary to the finding of Gaunt and of the HAPMAPlinkage analysis of this region, we found no evidence for strong linkagedisequilibrium. These findings indicate an unexpected frequency ofrecombination within this relatively small region of genomic sequence.These findings support the conclusion that for this locus, the use ofassays detecting more than one polymorphic marker which are collocatedon what were believed to be the same prior art “linkage block” willincrease the informativity of a test to determine imprinting status of asample. Panels of markers to be quantified may be based on combinationsof SNPs among the previously described SNPs and the novel SNPs describedin this application, and these findings demonstrate, counter to what wasknown prior to our study, that such combinations of markers can improvethe ability to monitor LOI of IGF2 by dramatically increasing thepercentage of populations that can be tested.

Example 4 Demonstration of Use of SNPs to Determine LOI Status of IGF2

The SNP corresponding to SEQ ID NO: 64 (rs680) falls within the targetrecognition sequences of two restriction enzymes, Apa I and Ava II.These two enzymes cleave in an allele-specific manner. Apa I recognizesand cleaves the sequence when the “G” allele is present, and Ava IIrecognizes and cleaves the sequence when the “A” allele is present. Toindependently assess genotypes within a selected panel of individuals, aPCR amplicon including the position of SEQ ID NO: 64 was amplified froma genomic DNA sample derived from each individual. Amplicons weredigested with Apa I or Ava II or a combination of both enzymes.Digestion by Apa I only indicates that the individual is homozygous forthe G allele, digestion by Ava II only indicates that the individual ishomozygous for the A allele, and digestion by both enzymes indicatesthat the individual is heterozygous for the SNP. An example of the dataoutput for each possible genotype of SEQ ID NO: 64 is shown in FIG. 5.As described above, the genotype call determined by the digestion-basedassay exactly matched the genotype call based on DNA sequencing in 70 of70 individuals (100%).

The same basic assay strategy can be utilized to detect LOI of IGF2,provided the individual being tested is heterozygous for SEQ ID NO: 64.An example is shown in FIG. 6. Total RNA was extracted from threeindividuals heterozygous for SEQ ID NO: 64. Two individuals werepreviously shown to be LOI for IGF2 and the third was previously shownto display normal imprinting of IGF2. The region including SEQ ID NO: 64was RT-PCR amplified from each sample. Reactions lacking reversetranscriptase were performed in parallel to confirm that there was noamplification from genomic DNA. RT-PCR amplicons were then digested withApa I or Ava II or a combination of both enzymes, as described above.Digested products were resolved on an Agilent Bioanalyzer, andconcentrations of cut and uncut fragments were determined. The quantityof fragments cut by Apa I represents the proportion of cDNA amplifiedfrom the “G” allele. The quantity of fragments cut by Ava II representsthe proportion of cDNA amplified from the “A” allele. Therefore, theratio of Apa I cut fragments to Ava II cut fragments indicates therelative ratio of expression of the two alleles in the original RNAsample. As shown in FIG. 6, Sample 2 expresses exclusively the “A”allele. Samples 1 and 3 express both alleles (i.e. display LOI IGF2),with G:A ratios of 0.5 and 0.3, respectively. As described above,previous studies have used a threshold of 33.3% expression from thelesser abundant allele relative to the more abundant allele as thedefinition for LOI of IGF2.

Other SNPs that can be useful for detecting LOI of IGF2 do not fallwithin restriction enzyme recognition sequences. Therefore, the abilityto monitor LOI in a given individual is improved by developingallele-specific gene expression assays that do not require restrictionenzyme digestion. As a demonstration, we developed a primer extensionbased assay for SEQ ID NO: 64. FIG. 7 diagrams the use of a primerextension assay for genotyping SEQ ID NO: 64. The region including theSNP of interest is PCR amplified using genomic DNA obtained from theindividual to be genotyped. A primer is added to the purified PCRproduct that anneals with its 3′ terminal nucleotide complimentary tothe template nucleotide 1 base to the 3′ side of the polymorphicnucleotide to be genotyped. Single nucleotide primer extension iscarried out using a thermostable DNA polymerase and differentiallyfluorescently labeled ddNTPs. In the example diagrammed in FIG. 7,either dR110 labeled ddGTP or dR6G labeled ddATP is added to the 3′ endof the primer. These labeled polynucleotides are then resolved and thepeak areas representative of each possible incorporated nucleotide arecalculated (i.e. using an ABI 3730 Gene Analyzer with Gene Mappersoftware). Peak areas are compared to determine the genotype of theindividual at that SNP position.

The three individuals that were assayed for LOI of IGF2 by therestriction enzyme based assay (FIG. 6) were genotyped for SEQ ID NO: 64using the primer extension assay (FIG. 8). As expected, peaksrepresenting both alleles of the SNP were detected, confirming that thethree individuals are heterozygous for SEQ ID NO: 64.

To measure allele-specific expression of IGF2 in the same threeindividuals, the region including SEQ ID NO: 64 was RT-PCR amplifiedfrom a total RNA sample derived from each individual. Reactions lackingreverse transcriptase were performed in parallel to confirm that therewas no amplification from genomic DNA. The cDNA products obtained werepurified and analyzed as diagrammed in FIG. 7. Peak areas representingeach of the two possible alleles were calculated. To correct fordifferences in dye intensities, these values were normalized based oncomparisons of peak areas calculated using predetermined 1:1 ratios ofeach allele (i.e. 1:1 ratio of DNA amplicons derived from individualsthat are homozygous for each of the two alleles). The resultingchromatograms and calculated allele ratios are shown in FIG. 9. Inagreement with the results shown in FIG. 6, Samples 1 and 3 weredetermined to show LOI of IGF2, and Sample 2 was determined to shownormal imprinting of IGF2. T4he same type of single nucleotide primerextension assay that utilizes any SNP within the transcribed region ofIGF2 could be used to monitor allele-specific expression of IGF2.

To demonstrate the use of additional SNPs for measuring allele-specificexpression of IGF2, single nucleotide primer extension assays weredesigned based on eight additional SNPs (SEQ ID NO: 1, 10, 21, 56, 83,85, 102 and 111). The SNPs corresponding to SEQ ID NO: 1, 10 and 21 arenovel SNPs. For each of the nine SNPs (including SEQ ID NO: 64), PCRproducts were separately amplified from genomic DNA samples derived fromtwo individuals; one homozygous for one allele of the SNP and the otherhomozygous for the other allele of the SNP. The PCR products werepurified and quantified. For each of the nine SNPs, two PCR products(one amplified from the DNA sample homozygous for one allele and theother amplified from the DNA sample homozygous for the other allele)were combined in the following ratios of allele 1 to allele 2; 1:10,1:8, 1:6, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 6:1, 8:1 and 10:1. For eachof the nine SNPs, the single nucleotide primer extension assay wasperformed in triplicate on each dilution point. Peak areas representingeach of the two possible alleles were calculated. To correct fordifferences in dye intensities, these values were normalized based oncomparisons of peak areas calculated using predetermined 1:1 ratios ofeach allele. The analytical quantitative linearity of each assay isshown in FIG. 10. The average R² of the assays is 0.996±0.002 and theaverage slope is 0.830±0.024.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

TABLE 1A Known Polymorphisms in IGF2 exons 8 & 9. Seq. Genomic IDPosition* dbSNP Alleles Block Sequence 48 2111395 rs1803647 [C/G] 1TGCTGTTTCCGCAGCTGTGACCTGGC[C/G]CTCCTGGAGACGTACTGTGCTACCC 49 2110976rs14367 [G/A] 1 AGTTCTTCCAATATGACACCTGGAA[G/A]CAGTCCACCCAGCGCCTGCGCAGGG50 2110955 rs1065443 [C/T] 1GGAAGCAGTCCACCCAGCGCCTGCG[C/T]AGGGGCCTGCCTGCCCTCCTGCGTG 51 2110819rs11545014 [C/T] 1AAGACCCCGCCCACGGGGGCGCCCCC[C/T]CAGAGATGGCCAGCAATCGGAAGTG 52 2110818rs1050342 [A/C] 1AGACCCCGCCCACGGGGGCGCCCCCC [A/C]AGAGATGGCCAGCAATCGGAAGTGA 53 2110796rs12993 [G/T] 1 CCCCCAGAGATGGCCAGCAATCGGAA[G/T]TGAGCAAAACTGCCGCRAGTCTGCA54 2110779 rs9282726 [A/G] 1CAATCGGAAKTGAGCAAAACTGCCGC[A/G]AGTCTGCAGCCYGGYGCCACCATCC 55 2110767rs3741214 [C/T] 1AGCAAAACTGCCGCAAGTCTGCAGCC[C/T]GGCGCCACCATCCTGCAGCCTCCTC 56 2110764rs2230949 [C/T] 1AAACTGCCGCAAGTCTGCAGCCCGG[C/T]GCCACCATCCTGCAGCCTCCTCCTG 57 2110733rs3213234 [G/T] 1CATCCTGCAGCCTCCTCCTGACCAC[G/T]GACGTTTCCATCAGGTTCCATCCCG 58 2110683rs34337549 [−/G] 1CGGGGACTGGGTCAGGAGAAGCCCCA[−/G]GGGGACGTGGAACCGAGAGATTTTC 59 2110613rs6223 [A/C] 1 CAGGCTACTCTCCTCGGCCCCCTCC [A/C]TCGGGCTGAGGAAGCACAGCAGCAT60 2110586 rs11510 [A/T] 1TCGGGCTGAGGAAGCACAGCAGCATC[A/T]TCAAACATGTACAAAATCGATTGGC 61 2110554rs1803648 [A/C] 1ATGTACAAAATCGATTGGCTTTAAA[A/C]ACCCTTCACATACCCTCCCCCCAAA 62 2110470rs11564731 [−/AC] 1AAAACATTAAACTAACCCCCTTCCCC [−/AC]CCCCCCCACAACAACCCTCTTAAAA 63 2110279rs15737 [C/T] 1 TGGCACTCCCCACCCCCCTCTTTCT[C/T]TTCTCCCTTGGACTTTGAGTCAAAT64 2110210 rs680 [G/A/C/T] 2CTGAACCAGCAAAGAGAAAAGAAGG[G/A/C/T]CCCCAGAAATCACAGGTGGGCACGT 65 2110180rs1065685 [C/G] 2CAGAAATCACAGGTGGGCACGTCGCT[C/G]CTACCGCCATCTCCCTTCTCACGGG 66 2109887rs56731553 [C/T] 2GTGCTCGTGTGTGTGCTGTGTTCATG[C/T]GTGTGCTGTGTGTTGTGTGTGTGTA 67 2109881rs56154171 [C/T] 2TGTGCTGTGCTCGTGTGTGTGCTGTG[C/T]TCATGCGTGTGCTGTGTGTTGTGTG 68 2109862rs59198946 [−/AT] 2GTGCTGTGCGTTTGTGTGTGTGCTGT [−/AT]GCTCGTGTGTGTGCTGTGTTCATGC 69 2109849rs61872709 [C/T] 2TCTGTGTGCTGTGTGTGCTGTGCGTT[C/T]GTGTGTGTGCTGTGCTCGTGTGTGT 70 2109847rs61872708 [C/T] 2CATCTGTGTGCTGTGTGTGCTGTGCG[C/T]TTGTGTGTGTGCTGTGCTCGTGTGT 71 2109730rs61872707 [A/G] 2TGCGTTTGTGTGTGTGCTGTGTGTGC[A/G]TGTGTGTGCGTGTGTGTGCCGTGCG 72 2109724rs61872706 [A/G] 2GTGCTGTGCGTTTGTGTGTGTGCTGT[A/G]TGTGCATGTGTGTGCGTGTGTGTGC 73 2109712rs11042774 [C/T] 2gtgegtttgtgtgtgctgtgegtttg [C/T]gtgtgtgctgtgtgtgcatgtgtgt 74 2109604rs59630895 [−/TGTG] 2TTGTGTGTGTGCTGTGTGCTAGTGTG[−/TGTG]CTGTGTGTGCATGTGTGTGCGTGTG 75 2109541rs7111331 [C/T] 2tgctgtgttcgtgtgtgctgtgttcg[C/T]gtgtgtgtgctgtgtgtgcatgtgt *Coordinatesrelative to NCBI Build 36, Chr: 11

TABLE 1B Known Polymorphisms in IGF2 exon 9. Seq. Genomic ID Position*dbSNP Alleles Block Sequence 76 2108682 rs11042767 [C/T] 3ACATTTCTTGGGGGGTCCCCAGGAGA[C/T]GGGCAAAGATGATCCCTAGGTGTGC 77 2108628rs7129583 [C/T] 3AGTCCTCGGGGGCCGTGCACTGATG[C/T]GGGGAGTGTGGGAAGTCTGGCGGTT 78 2108395rs28462050 [T/G] 3GCATTTTTCCTTTTTTTTTTTTTTT[T/G]GTTTTTTTTTTACCCCTCCTTAGCT 79 2108344rs28472590 [−/GG] 3TGCCCCCCTGTTACATGGGGGGGGGG[−/GG]TTTAATTTGGTTTCTGAGCGCATAA 80 2108288rs58312807 [C/T] 3GAGTCCTCGGGGGCCGTGCACTGATG[C/T]GGGGAGTGTGGGAAGTCTGGCGGTT 81 2107971rs60649995 [G/A] 3AGGCTGGCCGGAGGGGAAGGGGCTA[G/A]CAGGTGTGTAAACAGAGGGTTCCAT 82 2107909rs58562468 [A/G] 3CAGGGTGGCCGCCTTCCGCACACTTG[A/G]GGAACCCTCCCCTCTCCCTCGGTGA 83 2107900rs1065687 [C/G] 3CGCCTTCCGCACACTTGAGGAACCCT[C/G]CCCTCTCCCTCGGTGACATCTTGCC 84 2109167rs3208122 [A/C] 2TAAGCAACTACGATATCTGTATGGAT[A/C]AGGCCAAAGTCCCGCTAAGATTCTC 85 2109117rs3168310 [C/G] 2CCAATGTTTTCATGGTCTGAGCCCC[C/G]CTCCTGTTCCCATCTCCACTGCCCC 86 2108911rs58527086 [−/T] 3CATCGTGGCTCACGCTGCGGGGGCCG[−/T]GGGGACAGGCGCCAAGGAGGCCAGC 87 2108682rs57156844 [C/T] 3ACATTTCTTGGGGGGTCCCCAGGAGA[C/T]GGGCAAAGATGATCCCTAGGTGTGC 88 2108628rs3802971 [C/T] 3AGTCCTCGGGGGCCGTGCACTGATG[C/T]GGGGAGTGTGGGAAGTCTGGCGGTT 89 2108395rs3180700 [T/G] 3GCATTTTTCCTTTTTTTTTTTTTTT[T/G]GTTTTTTTTTTACCCCTCCTTAGCT 90 2108344rs57423851 [−/GG] 3TGCCCCCCTGTTACATGGGGGGGGGG[−/GG]TTTAATTTGGTTTCTGAGCGCATAA 91 2108288rs35818489 [C/T] 3GAGTCCTCGGGGGCCGTGCACTGATG[C/T]GGGGAGTGTGGGAAGTCTGGCGGTT 92 2107971rs11825733 [G/A] 3AGGCTGGCCGGAGGGGAAGGGGCTA[G/A]CAGGTGTGTAAACAGAGGGTTCCAT 93 2107909rs11541377 [A/G] 3CAGGGTGGCCGCCTTCCGCACACTTG[A/G]GGAACCCTCCCCTCTCCCTCGGTGA 94 2107900rs11541375 [C/G] 3CGCCTTCCGCACACTTGAGGAACCCT[C/G]CCCTCTCCCTCGGTGACATCTTGCC 95 2107862rs11541373 [C/T] 3GGTGACATCTTGCCCGCCCCTCAGCA[C/T]CCTGCCTTGTCTCCAGGAGGTCCGA 96 2107847rs11541372 [A/C] 3CCCCTCAGCACCCTGCCTTGTCTCC[A/C]GGAGGTCCGAAGCTCTGTGGGACCT 97 2107755rs11541374 [G/T] 3CAGGCGGGTCTGAGCCCACAGAGCAG[G/T]AGAGCTGCCAGGTCTGCCCATCGAC 98 2107602rs3189464 [A/C] 3CCTCGCCCCCACTTGTGCCCCCAGCT[A/C]AGCCCCCCTGCACGCAGCCCGACTA 99 2107472rs61745040 [C/T] 3CAGTCGCAGAGGGTCCCTCGGCAAG[C/T]GCCCTGTGAGTGGGCCATTCGGAAC 100  2107471rs11564732 [G/A/T] 3AGTCGCAGAGGGTCCCTCGGCAAGC[G/A/T]CCCTGTGAGTGGGCCATTCGGAACA *Coordinatesrelative to NCBI Build 36, Chr: 11

TABLE 1C Known Polymorphisms in IGF2 exon 9. Seq. Genomic ID Position*dbSNP Alleles Block Sequence 101 2107452 rs11541376 [C/T] 3GCAAGCGCCCTGTGAGTGGGCCATT[C/T]GGAACATTGGACAGAAGCCCAAAGA 102 2107273rs7873 [A/G] 3 GTGTTCCCGGGGGCACTTGCCGACC[A/G]GCCCCTTGCGTCCCCAGGTTTGCAG103 2107263 rs61745039 [G/A/T] 3GGGCACTTGCCGACCAGCCCCTTGC[G/A/T]TCCCCAGGTTTGCAGCTCTCCCCTG 104 2107147rs3177805 [C/T] 3TTGTCTCCTCCCCGTGTCCCCAATGT[C/T]TTCAGTGGGGGGCCCCCTCTTGGGT 105 2107135rs1065715 [C/G] 3CGTGTCCCCAATGTCTTCAGTGGGGG[C/G]CCCCCTCTTGGGTCCCCTCCTCTGC 106 2107134rs11541371 [C/G] 3TGTCCCCAATGTCTTCAGTGGGGGG[C/G]CCCCTCTTGGGTCCCCTCCTCTGCC 107 2107128rs1049926 [C/T] 3CCAATGTCTTCAGTGGGGGGCCCCCT[C/T]TTGGGTCCCCTCCTCTGCCATCACC 108 2107113rs3177946 [C/T] 3GGGGCCCCCTCTTGGGTCCCCTCCT[C/T]TGCCATCACCTGAAGACCCCCACGC 109 2107049rs1050035 [A/C] 3GTCACCTGTGCCTGCCGCCTCGGTCC[A/C]CCTTGCGGCCCGTGTTTGACTCAAC 110 2107027rs11541370 [A/G] 3AATATTAGCGTTAAAGGAGCTGAGTT[A/G]AGTCAAACACGGGCCGCAAGGTGGA 111 2107020rs2585 [G/A/C/T] 3TGCGGCCCGTGTTTGACTCAACTCA[G/A/C/T]CTCCTTTAACGCTAATATTTCCGGC 112 2106955rs1050141 [C/A] 3GGGTTTTGTCTTTAACCTTGTAACG[C/A]TTGCAATCCCAATAAAGCATTAAAA *Coordinatesrelative to NCBI Build 36, Chr: 11

TABLE 2A Novel Polymorphisms in IGF2 exon 9. Genomic Seq. ID Position*Alleles Block Sequence  1 2110869 [G/A] 1GCGTTCAGGGAGGCCAAACGTCACC[G/A]TCCCCTGATTGCTCTACCCACCCAA  2 2110827 [G/A]1 CCCACCCAAGACCCCGCCCACGGGG[G/A]CGCCCCCCCAGAGATGGCCAGCAAT  3 2110825[G/A] 1 CACCCAAGACCCCGCCCACGGGGGC[G/A]CCCCCCCAGAGATGGCCAGCAATCG  42110781 [G/A] 1 GCAATCGGAAGTGAGCAAAACTGCC[G/A]CAAGTCTGCAGCCCGGCGCCACCAT 5 2110657 [G/A] 1TGGGGCTTCTCCTGACCCAGTCCCC[G/A]TGCCCCGCCTCCCCGAAACAGGCTA  6 2110465 [C/T]1 TTAAACTAACCCCCTTCCCCCCCCC[C/T]CACAACAACCCTCTTAAAACTAATT  7 2110430[G/A/T] 1 CCTCTTAAAACTAATTGGCTTTTTA[G/A/T]AAACACCCCACAAAAGCTCAGAAAT  82110287 [C/G] 1 AAGGAATTTGGCACTCCCCACCCCC[C/G]TCTTTCTCTTCTCCCTTGGACTTTG 9 2110197 [C/T] 2GAGAAAAGAAGGACCCCAGAAATCA[C/T]AGGTGGGCACGTCGCTGCTACCGCC 10 2110187 [C/T]2 GGACCCCAGAAATCACAGGTGGGCA[C/T]GTCGCTGCTACCGCCATCTCCCTTC 11 2110129[G/A] 2 AATTTTCAGGGTAAACTGGCCATCC[G/A]AAAATAGCAACAACCCAGACTGGCT 122110109 [C/T] 2 CATCCGAAAATAGCAACAACCCAGA[C/T]TGGCTCCTCACTCCCTTTTCCATCA13 2110063 [A/C] 2CATCACTAAAAATCACAGAGCAGTC[A/C]GAGGGACCCAGTAAGACCAAAGGAG 14 2110060 [G/C]2 CACTAAAAATCACAGAGCAGTCAGA[G/C]GGACCCAGTAAGACCAAAGGAGGGG 15 2110058[G/T/A] 2 CTAAAAATCACAGAGCAGTCAGAGG[G/T/A]ACCCAGTAAGACCAAAGGAGGGGAG 162109220 [A/C] 2 GCGCACACACACGCACACCCCCACA[A/C]AATTGGATGAAAACAATAAGCATAT17 2109153 [G/A] 2TCTGTATGGATCAGGCCAAAGTCCC[G/A]CTAAGATTCTCCAATGTTTTCATGG 18 2109095 [C/T]3 CCCGCTCCTGTTCCCATCTCCACTG[C/T]CCCTCGGCCCTGTCTGTGCCCTGCC 19 2109074[C/G] 3 ACTGCCCCTCGGCCCTGTCTGTGCC[C/G]TGCCTCTCAGAGGAGGGGGCTCAGA 202108843 [T/C] 3 CATTCCCGATACACCTTACTTACTG[T/C]GTGTTGGCCCAGCCAGAGTGAGGAA21 2108835 [C/T] 3ATACACCTTACTTACTGTGTGTTGG[C/T]CCAGCCAGAGTGAGGAAGGAGTTTG 22 2108806[A/C/T] 3 GCCAGAGTGAGGAAGGAGTTTGGCC[A/C/T]CATTGGAGATGGCGGTAGCTGAGCA 232108738 [G/A/T] 3AGCCTGACTCCCTGGTGTGCTCCTG[G/A/T]AAGGAAGATCTTGGGGACCCCCCCA 24 2108440[C/T/G] 3 CAAATTTCATGTCAATTGATCTATT[C/T/G]CCCCTCTTTGTTTCTTGGGGCATTT 252108424 [T/G] 3 TGATCTATTCCCCCTCTTTGTTTCT[T/G]GGGGCATTTTTCCTTTTTTTTTTTT26 2108417 [T/G] 3TTCCCCCTCTTTGTTTCTTGGGGCA[T/G]TTTTCCTTTTTTTTTTTTTTTTGTT 27 2108326 [G/A]3 AATTAAACCCCCCCCCCATGTAACA[G/A]GGGGGCAGTGACAAAAGCAAGAACG 28 2107988[C/T/A] 3 GGCTCCTGGCTGGCCTGAGGCTGGC[C/T/A]GGAGGGGAAGGGGCTAGCAGGTGTG 292107918 [G/C/A] 3GGCTGGGGCAGGGTGGCCGCCTTCC[G/C/A]CACACTTGAGGAACCCTCCCCTCTC *Coordinatesrelative to NCBI Build 36, Chr: 11

TABLE 2B Novel Polymorphisms in IGF2 exon 9. Genomic Seq. ID Position*Alleles Block Sequence 30 2107819 [T/G] 3AGGTCCGAAGCTCTGTGGGACCTCT[T/G]GGGGGCAAGGTGGGGTGAGGCCGGG 31 2107776 [G/A]3 AGGCCGGGGAGTAGGGAGGTCAGGC[G/A]GGTCTGAGCCCACAGAGCAGGAGAG 32 2107668[G/A] 3 ATGCCATAGCAGCCACCACCGCGGC[G/A]CCTAGGGCTGCGGCAGGGACTCGGC 332107664 [A/T] 3 CATAGCAGCCACCACCGCGGCGCCT[A/T]GGGCTGCGGCAGGGACTCGGCCTCT34 2107625 [C/T] 3GACTCGGCCTCTGGGAGGTTTACCT[C/T]GCCCCCACTTGTGCCCCCAGCTCAG 35 2107595 [C/G]3 CCACTTGTGCCCCCAGCTCAGCCCC[C/G]CTGCACGCAGCCCGACTAGCAGTCT 36 2107523[C/T] 3 CCTGGTGACGGGGCTGGCATGACCC[C/T]GGGGGTCGTCCATGCCAGTCCGCCT 372107478 [G/A] 3 CCGCCTCAGTCGCAGAGGGTCCCTC[G/A]GCAAGCGCCCTGTGAGTGGGCCATT38 2107472 [C/T] 3CAGTCGCAGAGGGTCCCTCGGCAAG[C/T]GCCCTGTGAGTGGGCCATTCGGAAC 39 2107469 [C/T]3 TCGCAGAGGGTCCCTCGGCAAGCGC[C/T]CTGTGAGTGGGCCATTCGGAACATT 40 2107379[C/T] 3 ACCCACATTGGCCTGAGATCCAAAA[C/T]GCTTCGAGGCACCCCAAATTACCTG 412107353 [C/G] 3 GCTTCGAGGCACCCCAAATTACCTG[C/G]CCATTCGTCAGGACACCCACCCACC42 2107278 [C/T] 3AGTGGGTGTTCCCGGGGGCACTTGC[C/T]GACCAGCCCCTTGCGTCCCCAGGTT 43 2107263 [G/A]3 GGGCACTTGCCGACCAGCCCCTTGC[G/A]TCCCCAGGTTTGCAGCTCTCCCCTG 44 2107151[A/G] 3 ATCTTGTCTCCTCCCCGTGTCCCCA[A/G]TGTCTTCAGTGGGGGGCCCCCTCTT 452107054 [G/A] 3 GAATGTCACCTGTGCCTGCCGCCTC[G/A]GTCCACCTTGCGGCCCGTGTTTGAC46 2107037 [G/A] 3GCCGCCTCGGTCCACCTTGCGGCCC[G/A]TGTTTGACTCAACTCAACTCCTTTA 47 2106956[G/A/C] 3 TGGGTTTTGTCTTTAACCTTGTAAC[G/A/C]CTTGCAATCCCAATAAAGCATTAAA*Coordinates relative to NCBI Build 36,Chr: 11

TABLE 3 Observed Heterozygosity of Transcribed IGF2 SNPs in HumanPopulations SEQ ID Genomic NO: Position Block All ObsHET AA ObsHET CAUObsHET 1 2110869 1 1.08% (6 of 553) 3.3% (3 of 91) 1% (2 of 201) 42110781 1 0.95% (4 of 421) 2.9% (2 of 69) 0% (0 of 155) 56 2110764 11.45% (1 of 69) 6.25% (1 of 16) 0% (0 of 35) 6 2110465 1 3.38% (7 of207) 11.48% (7 of 61) 0% (0 of 51) 7 2110430 1 1.23% (1 of 81) 16.67% (1of 6) 0% (0 of 54) 64 2110210 2 54.46% (110 of 202) 13.89% (5 of 36)63.08% (41 of 65) 10 2110187 2 2.82% (15 of 531) 17.33% (13 of 75) 1% (2of 201) 16 2109220 2 1.57% (9 of 572) 9.88% (8 of 81) 0% (0 of 214) 832109215 2 3.28% (19 of 579) 0% (0 of 94) 8.13% (17 of 209) 85 2109117 238.59% (137 of 355) 27.27% (18 of 66) 40.16% (49 of 122) 20 2108843 30.35% (2 of 566) 2.3% (2 of 87) 0% (0 of 202) 21 2108835 3 4.07% (20 of492) 1.43% (1 of 70) 9.94% (18 of 181) 87 2108682 3 1.89% (5 of 265)14.29% (4 of 28) 1.01% (1 of 99) 88 2108628 3 7.57% (14 of 185) 3.13% (1of 32) 4.88% (2 of 41) 25 2108424 3 0.35% (1 of 288) 2.17% (1 of 46) 0%(0 of 89) 26 2108417 3 3.38% (9 of 266) 7.32% (3 of 41) 6.74% (6 of 89)92 2107971 3 2.8% (5 of 536) 16.3% (15 of 92) 0% (0 of 185) 32 2107668 32.35% (12 of 511) 0% (0 of 94) 0.7% (1 of 142) 100 2107471 3 4.66% (26of 558) 1.05% (1 of 95) 1.97% (4 of 203) 102 2107273 3 6.41% (32 of 499)14.81% (8 of 54) 9.34% (17 of 182) 111 2107020 3 50.19% (135 of 269)23.08% (3 of 13) 49.58% (59 of 119) 47 2106956 3 0.33% (1 of 304) 0% (0of 70) 0% (0 of 115) SEQ ID NO: CHI ObsHET JAP ObsHET MEX ObsHET 1 0% (0of 84) 0% (0 of 90) 1.15% (1 of 87) 4 0% (0 of 61) 2.86% (2 of 70) 0% (0of 66) 56 0% (0 of 8) 0% (0 of 2) 0% (0 of 8) 6 0% (0 of 21) 0% (0 of29) 0% (0 of 45) 7 0% (0 of 8) 0% (0 of 7) 0% (0 of 6) 64 70.83% (17 of24) 64.1% (25 of 39) 57.89% (22 of 38) 10 0% (0 of 80) 0% (0 of 89) 0%(0 of 86) 16 0% (0 of 90) 0% (0 of 91) 1.04% (1 of 96) 83 0% (0 of 90)0% (0 of 91) 2.11% (2 of 95) 85 40.74% (22 of 54) 53.85% (35 of 65)27.08% (13 of 48) 20 0% (0 of 90) 0% (0 of 91) 0% (0 of 96) 21 0% (0 of79) 0% (0 of 81) 1.23% (1 of 81) 87 0% (0 of 41) 0% (0 of 52) 0% (0 of45) 88 12.2% (5 of 41) 20% (5 of 25) 2.17% (1 of 46) 25 0% (0 of 67) 0%(0 of 57) 0% (0 of 29) 26 0% (0 of 44) 0% (0 of 56) 0% (0 of 36) 92 0%(0 of 79) 0% (0 of 87) 0% (0 of 93) 32 10.23% (9 of 88) 2.2% (2 of 91)0% (0 of 96) 100 1.16% (1 of 86) 5.75% (5 of 87) 17.24% (15 of 87) 1020% (0 of 86) 0% (0 of 85) 7.61% (7 of 92) 111 50% (24 of 48) 53.85% (21of 39) 56% (28 of 50) 47 0% (0 of 43) 0% (0 of 42) 2.94% (1 of 34) *Coordinates relative to NCBI Build 36, Chr: 11

TABLE 4 Informative SNPs in Chinese SEQ Genomic ID NO: Position BlockObs Het 64 2110210 2 70.83% (17 of 24) 85 2109117 2 40.74% (22 of 54) 882108628 3 12.2% (5 of 41) 32 2107668 3 10.23% (9 of 88) 111 2107020 350% (24 of 48)

TABLE 5 Informative SNPs in Japanese SEQ ID Genomic NO: Position BlockObs Het 4 2110781 1 2.86% (2 of 70) 64 2110210 2 64.1% (25 of 39) 852109117 2 53.85% (35 of 65) 88 2108628 3 20% (5 of 25) 32 2107668 3 2.2%(2 of 91) 100 2107471 3 5.75% (5 of 87) 111 2107020 3 53.85% (21 of 39)

TABLE 6 Inf. SNPs in African Amer. SEQ Genomic ID NO: Position Block ObsHet 1 2110869 1 3.3% (3 of 91) 4 2110781 1 2.9% (2 of 69) 56 2110764 16.25% (1 of 16) 6 2110465 1 11.48% (7 of 61) 7 2110430 1 16.67% (1 of 6)64 2110210 2 13.89% (5 of 36) 10 2110187 2 17.33% (13 of 75) 16 21092202 9.88% (8 of 81) 85 2109117 2 27.27% (18 of 66) 20 2108843 3 2.3% (2 of87) 87 2108682 3 14.29% (4 of 28) 88 2108628 3 3.13% (1 of 32) 252108424 3 2.17% (1 of 46) 26 2108417 3 7.32% (3 of 41) 92 2107971 316.3% (15 of 92) 102 2107273 3 14.81% (8 of 54) 111 2107020 3 23.08% (3of 13)

TABLE 7 Informative SNPs in Caucasian SEQ Genomic Block Obs Het 642110210 2 63.08% (41 of 65) 83 2109215 2 8.13% (17 of 209) 85 2109117 240.16% (49 of 122) 21 2108835 3 9.94% (18 of 181) 88 2108628 3 4.88% (2of 41) 26 2108417 3 6.74% (6 of 89) 100 2107471 3 1.97% (4 of 203) 1022107273 3 9.34% (17 of 182) 111 2107020 3 49.58% (59 of 119)

TABLE 8 Informative SNPs in Mexicans SEQ ID Genomic Block Obs Het 642110210 2 57.89% (22 of 38) 83 2109215 2 2.11% (2 of 95) 85 2109117 227.08% (13 of 48) 88 2108628 3 2.17% (1 of 46) 100 2107471 3 17.24% (15of 87) 102 2107273 3 7.61% (7 of 92) 111 2107020 3 56% (28 of 50) 472106956 3 2.94% (1 of 34)

What is claimed is:
 1. A method of quantifying allelic-specificexpression of RNA in a human individual that is a heterozygote for asingle nucleotide polymorphism (SNP) in the Insulin Growth Factor-2(IGF2) gene, the method comprising quantifying in a sample from theindividual the amount of RNA comprising each polymorphic option of theSNP, wherein the SNP comprises SEQ ID NO:32.
 2. The method of claim 1,wherein the sample is a blood sample.
 3. The method of claim 1, whereinthe sample is a stool or tissue sample.
 4. The method of claim 1,wherein the quantifying comprises reverse transcribing RNA from theindividual into cDNA and determining the amount of RNA based upon thequantity of allele-specific IGF2 cDNA resulting from the reversetranscribing.
 5. The method of claim 4, wherein the quantifyingcomprises amplifying the IGF2 cDNA or a portion thereof comprising atleast one of the polymorphic options.
 6. The method of claim 4, whereinthe quantifying comprises nucleotide sequencing the IGF2 cDNA, or aportion thereof comprising at least one of the polymorphic options, fromthe individual.
 7. The method of claim 4, wherein the quantifyingcomprises contacting the IGF2 cDNA, or a portion thereof comprising atleast one of the polymorphic options, from the individual with anoligonucleotide that distinguishes between polymorphic options of theSNP.
 8. The method of claim 7, wherein the penultimate or ultimate 3′nucleotide of the oligonucleotide hybridizes to the polymorphicnucleotide of the SNP.
 9. The method of claim 4, wherein the quantifyingcomprises contacting the IGF2 cDNA, or a portion thereof comprising atleast one of the polymorphic options, from the individual with anoligonucleotide that is complementary to the nucleotide immediatelyupstream of the polymorphic nucleotide of the SNP.
 10. The method ofclaim 1, wherein the quantifying comprises nucleotide sequencing theRNA, or a portion thereof comprising at least one of the polymorphicoptions, from the individual.
 11. The method of claim 1, wherein thequantifying comprises contacting the RNA from the individual with anoligonucleotide that distinguishes between polymorphic options of theSNP.
 12. The method of claim 11, wherein the penultimate or ultimate 3′nucleotide of the oligonucleotide hybridizes to the polymorphicnucleotide of the SNP.
 13. The method of claim 1, wherein thequantifying comprises contacting the RNA, or a portion thereofcomprising at least one of the polymorphic options, from the individualwith an oligonucleotide that is complementary to the nucleotideimmediately upstream of the polymorphic nucleotide of the SNP.
 14. Themethod of claim 1, wherein the quantifying comprises performingallele-specific hybridization, allele-specific amplification,restriction fragment length polymorphism analysis, denaturing gradientgel electrophoresis, or single-strand conformation polymorphismanalysis.
 15. The method of claim 1, further comprising obtaining asample comprising DNA from the individual and determining from the DNAfrom the sample that the individual is heterozygous for the SNP.
 16. Themethod of claim 1, wherein the individual is determined to have loss ofimprinting of IGF-2, and wherein the method further comprisesadministering an anti-cancer treatment to the individual.
 17. The methodof claim 1, wherein the individual is determined to have loss ofimprinting of IGF-2, and wherein the method further comprises performingadditional cancer diagnostic testing on the individual.